The isolation and survival characterization of radiation and chemical-mutagen sensitive mutants of Pseudomonas aeruginosa

Chlorate-resistant mutants of Arabidopsis thaliana were isolated in order to find nitrate reductase-less mutants. It appeared that chlorate resistance in higher plants can arise by mutations concerning two different mechanisms: (1) a lower reduction rate of chlorate due to a lower level of nitrate reductase activity; (2) a lower increase in content of chlorate and/or chlorite and of chloride after chlorate treatment. One mutant of the first type and two mutants of the second type are described. The nitrate reductase-less mutant grows poorly on a medium with nitrate as the only nitrogen source but is not blocked in the uptake of nitrate. Both the other mutants exhibit a nitrate reductase activity equal to or higher than that of the wild type, but probably have a much lowered uptake of chlorate. The latter two mutants belong to the same complementation group, whereas the nitrate reductase-less mutant belongs to a different group.     

Disruption of the nitrate transporter genes<Emphasis Type="Italic"> AtNRT2.1</Emphasis> and<Emphasis Type="Italic"> AtNRT2.2</Emphasis> restricts growth at low external nitrate concentration

The high-affinity transport systems in Arabidopsis thaliana (L.) Heynh. involve potentially seven genes. Among these, the AtNRT2.1 and/or AtNRT2.2 genes have been shown to play a major role in the inducible component of this transport system. The physiological impact of a disruption of AtNRT2.1 and AtNRT2.2 on plant growth and N-metabolism was investigated. The reduced nitrate uptake in the mutant under a limiting N-regime was found to correlate with a significant difference in shoot/root ratio between wild type and mutant and a drastically reduced nitrate level in the shoot of the mutant. Carbohydrate analyses of plants under a low nitrate supply revealed a slight increase in glucose and fructose in the mutant shoots as well as an increase in sucrose and starch contents in mutant shoots. Interestingly, the AtNRT2.4 and AtNRT2.5 genes were over-expressed in the mutant growing in reduced N-conditions, without any compensation by root nitrate influx. These results are discussed in the context of the putative role of the different NRT2 genes.Abbreviations DW Dry weight - FW Fresh weight - HATS High-affinity transport system - LATS Low-affinity transport system - NRT Nitrate transporter - WT Wild type     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Enhancement of cadmium accumulation in sweet sorghum as affected by nitrate

• The Cadmium (Cd)‐polluted soils are is an increasing concern worldwide. Phytoextraction of Cd pollutants by high biomass plants, such as sweet sorghum, is considered an environmentally‐friendly, cost‐effective and sustainable strategy for remediating this problem. Nitrogen (N) is a macronutrient essential for plant growth, development and stress resistance. Nevertheless, how nitrate, as an important form of N, affects Cd uptake, translocation and accumulation in sweet sorghum is still unclear.
• In the present study, a series of nitrate levels (N1, 0.5 mm ; N2, 2 mm ; N3, 4 mm ; N4, 8 mm and N5, 16 mm ) with or without added 5 μm CdCl₂ treatment in sweet sorghum was investigated hydroponically.
• The results indicate that Cd accumulation in the aboveground parts of sweet sorghum was enhanced by optimum nitrate supply, resulting from both increased dry weight and Cd concentration. Although root‐to‐shoot Cd translocation was not enhanced by increased nitrate, some Cd was transferred from cell walls to vacuoles in leaves. Intriguingly, expression levels of Cd uptake and transport genes, SbNramp1, SbNramp5 and SbHMA3, were not closely related to increased Cd as affected by nitrate supply. The expression of SbNRT1.1B in relation to nitrate transport showed an inverted ‘U’ shape with increasing nitrate levels under Cd stress, which was in agreement with trends in Cd concentration changes in aboveground tissues.
• Based on the aforementioned results, nitrate might regulate Cd uptake and accumulation through expression of SbNRT1.1B rather than SbNramp1, SbNramp5 or SbHMA3, the well‐documented genes related to Cd uptake and transport in sweet sorghum.

     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Abscisic acid signaling negatively regulates nitrate uptake via phosphorylation of NRT1.1 by SnRK2s in Arabidopsis

Nitrogen (N) is a limiting nutrient for plant growth and productivity. The phytohormone abscisic acid (ABA) has been suggested to play a vital role in nitrate uptake in fluctuating N environments. However, the molecular mechanisms underlying the involvement of ABA in N deficiency responses are largely unknown. In this study, we demonstrated that ABA signaling components, particularly the three subclass III SUCROSE NON‐FERMENTING1 (SNF1)‐RELATED PROTEIN KINASE 2S (SnRK2) proteins, function in root foraging and uptake of nitrate under N deficiency in Arabidopsis thaliana. The snrk2.2snrk2.3snrk2.6 triple mutant grew a longer primary root and had a higher rate of nitrate influx and accumulation compared with wild‐type plants under nitrate deficiency. Strikingly, SnRK2.2/2.3/2.6 proteins interacted with and phosphorylated the nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) in vitro and in vivo. The phosphorylation of NRT1.1 by SnRK2s resulted in a significant decrease of nitrate uptake and impairment of root growth. Moreover, we identified NRT1.1^Ser585 as a previously unknown functional site: the phosphomimetic NRT1.1^S585D was impaired in both low‐ and high‐affinity transport activities. Taken together, our findings provide new insight into how plants fine‐tune growth via ABA signaling under N deficiency.     

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.     

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.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron‐molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss‐of‐function mot1.2‐1 mutant showed reduced growth compared with wild‐type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum‐dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen‐fixing nodules, since genetic complementation with a wild‐type MtMOT1.2 gene or molybdate‐fortification of the nutrient solution, both restored wild‐type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.     

Lotus SHAGGY‐like kinase 1 is required to suppress nodulation in Lotus japonicus

Glycogen synthase kinase/SHAGGY‐like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell‐differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen‐fixing bacterium Mesorhizobium loti. RNAi knock‐down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy‐root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock‐down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON‐related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.     

CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots.

The Arabidopsis CHL1 (AtNRT1) gene confers sensitivity to the herbicide chlorate and encodes a nitrate-regulated nitrate transporter. However, how CHL1 participates in nitrate uptake in plants is not yet clear. In this study, we examined the in vivo function of CHL1 with in vivo uptake measurements and in situ hybridization experiments. Under most conditions tested, the amount of nitrate uptake by a chl1 deletion mutant was found to be significantly less than that of the wild type. This uptake deficiency was reversed when a CHL1 cDNA clone driven by the cauliflower mosaic virus 35S promoter was expressed in transgenic chl1 plants. Furthermore, tissue-specific expression patterns showed that near the root tip, CHL1 mRNA is found primarily in the epidermis, but further from the root tip, the mRNA is found in the cortex or endodermis. These results are consistent with the involvement of CHL1 in nitrate uptake at different stages of root cell development. A functional analysis in Xenopus oocytes indicated that CHL1 is a low-affinity nitrate transporter with a K(m) value of approximately 8.5 mM for nitrate. This finding is consistent with the chlorate resistance phenotype of chl1 mutants. However, these results do not fit the current model of a single, constitutive component for the low-affinity uptake system. To reconcile this discrepancy and the complex uptake behavior observed, we propose a "two-gene" model for the low-affinity nitrate uptake system of Arabidopsis.     

The soybean (Glycine max) nodulation‐suppressive CLE peptide,GmRIC1, functions interspecifically in common white bean (Phaseolus vulgaris), but not in a supernodulating line mutated in the receptor PvNARK

Legume plants regulate the number of nitrogen‐fixing root nodules they form via a process called the Autoregulation of Nodulation (AON). Despite being one of the most economically important and abundantly consumed legumes, little is known about the AON pathway of common bean (Phaseolus vulgaris). We used comparative‐ and functional‐genomic approaches to identify central components in the AON pathway of common bean. This includes identifying PvNARK, which encodes a LRR receptor kinase that acts to regulate root nodule numbers. A novel, truncated version of the gene was identified directly upstream of PvNARK, similar to Medicago truncatula, but not seen in Lotus japonicus or soybean. Two mutant alleles of PvNARK were identified that cause a classic shoot‐controlled and nitrate‐tolerant supernodulation phenotype. Homeologous over‐expression of the nodulation‐suppressive CLE peptide‐encoding soybean gene, GmRIC1, abolished nodulation in wild‐type bean, but had no discernible effect on PvNARK‐mutant plants. This demonstrates that soybean GmRIC1 can function interspecifically in bean, acting in a PvNARK‐dependent manner. Identification of bean PvRIC1, PvRIC2 and PvNIC1, orthologues of the soybean nodulation‐suppressive CLE peptides, revealed a high degree of conservation, particularly in the CLE domain. Overall, our work identified four new components of bean nodulation control and a truncated copy of PvNARK, discovered the mutation responsible for two supernodulating bean mutants and demonstrated that soybean GmRIC1 can function in the AON pathway of bean.     

Multiple roles of nitrate transport accessory protein NAR2 in plants

Two component high affinity nitrate transport system, NAR2/NRT2, has been defined in several plant species. In Arabidopsis, AtNAR2.1 has a role in the targeting of AtNRT2.1 to the plasma membrane. The gene knock out mutant atnar2.1 lacks inducible high-affinity transport system (IHATS) activity, it also shows the same inhibition of lateral root (LR) initiation on the newly developed primary roots as the atnrt2.1 mutant in response to low nitrate supply. In rice, OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a to provide nitrate uptake over high and low concentration ranges. In rice roots OsNAR2.1 and its partner NRT2s show some expression differences in both tissue specificity and abundance. It can be predicted that NAR2 plays multiple roles in addition to being an IHATS component in plants.Key words: NAR2, NRT2, nitrate transporter, root     

Alleviation of nitrate inhibition of soybean nodulation by high inoculum does not involve bacterial nitrate metabolism

Inoculation of soybean (Glycine max. cv. Bragg) plants with high level inoculum partially alleviated the nitrate inhibition of nodule formation (3 to 4 fold), but not nodule growth. This alleviation did not require the bacterial nitrate reductase asBradyrhizobium japonicum mutant strains 110CR1 and 110CR2 (both lacking assimilatory nitrate reductase activity) gave the same results as the wild type parent 311b110. The study was carried out in the glasshouse, thereby confirming preliminary field data by Herridgeet al. (1984) using a wild type bacterial inoculant.