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.     

Identification of an Arabidopsis solute carrier critical for intracellular transport and inter-organ allocation of molybdate

Plants represent an important source of molybdenum in the human diet. Recently, MOT1 has been identified as a transport protein responsible for molybdate import in Arabidopsis thaliana L.; however, the function of the homologous protein MOT2 has not been resolved. Interestingly, MOT2‐GFP analysis indicated a vacuolar location of this carrier protein. By site directed mutagenesis at the N‐terminal end of MOT2, we identified a di‐leucine motif that is essential for driving the protein into the vacuolar membrane. Molybdate quantification in isolated vacuoles showed that this organelle serves as an important molybdate store in Arabidopsis cells. When grown on soil, leaves from mot2 T‐DNA mutants contained more molybdate, whereas mot2 seeds contained significantly less molybdate than corresponding wild‐type (Wt) tissues. Remarkably, MOT2 mRNA accumulates in senescing leaves and mot2 leaves from plants that had finished their life cycle had 15‐fold higher molybdate levels than Wt leaves. Reintroduction of the endogenous MOT2 gene led to a Wt molybdate phenotype. Thus, mot2 mutants exhibit impaired inter‐organ molybdate allocation. As total concentrations of the molybdenum cofactor (Moco) and its precursor MPT correlates with leaf molybdate levels, we present novel evidence for an adjustment of Moco biosynthesis in response to cellular MoO₄^2? levels. We conclude that MOT2 is important for vacuolar molybdate export, an N‐terminal di‐leucine motif is critical for correct subcellular localisation of MOT2 and activity of this carrier is required for accumulation of molybdate in Arabidopsis seeds. MOT2 is a novel element in inter‐organ translocation of an essential metal ion.     

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.     

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.     

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.     

Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase

Summary Chlorate-resistant cell lines were established from survivors after plating allodihaploid cells of Nicotiana tabacum into solid medium containing 20 mM chlorate and amino acids as sole nitrogen source. Data characterizing 9 of the most resistant lines are presented. The mutational origin of these lines was inferred on the basis of the enhancement of the variant frequency by mutagen treatment, and of the persistance of the variant phenotype in cell progeny during growth in the absence of selection for more than 3 years and in plants regenerated from two of the lines.Seven lines completely lacked in vivo nitrate reductase (NR) activity and two lines exhibited low (less than 5% of the wild type) NR activity. The abolition of NR activity was found to be not due to an impaired induction by nitrate. Data reported elsewhere show that one of the NR-negative mutants simultaneously lacks xanthine dehydrogenase activity. This pleiotropic mutation is interpreted to affect the synthesis of a molybdenum-containing cofactor, whereas the 8 other lines carry mutations specifically affecting the synthesis of the NR. Both types of NR-negative mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They proved extremely sensitive to the standard medium containing nitrate and ammonium. Differences between the NR-negative mutants with respect to chlorate resistance suggest that chlorate inhibits cultured N tabacum cells not only via its NR-catalysed conversion to chlorite, but also by NR-independent mechanisms.     

Molybdenum Metabolism in Plants

Abstract: Among the micronutrients essential for plant growth and for microsymbionts, Mo is required in minute amounts. However, since Mo is often sequestered by Fe- or Al-oxihydrox-ides, especially in acidic soils, the concentration of the water-soluble molybdate anion available for uptake by plants may be limiting for the plant, even when the total Mo content of the soil is sufficient. In contrast to bacteria, no specific molybdenum uptake system is known for plants, but since molybdate and sulfate behave similarly and have similar structure, uptake of molybdate could be mediated unspecifically by one of the sulfate transporters. Transport into the different plant organs proceeds via xylem and phloem. A pterin-bound molybdenum is the cofactor of important plant enzymes involved in redox processes: nitrate reductase, xanthine dehydrogenase, aIdehyde oxidase, and probably sulfite oxidase. Biosynthesis of the molybdenum cofactor (Moco) starts with a guanosine-X-phos-phate. Subsequently, a sulfur-free pterin is synthesized, sulfur is added, and finally molybdenum is incorporated. In addition to the molybdopterin enzymes, small molybdopterin binding proteins without catalytic function are known and are probably involved in the storage of Moco. In symbiotic systems the nitrogen supply of the host plant is strongly influenced by the availability of Mo in soil, since both bacterial nitrogenase and NADPH-dependent nitrate reductase of mycorrhizal fungi are Mo enzymes.     

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.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Molecular mapping of the cnx2 locus involved in molybdenum cofactor biosynthesis in rice (Oryza sativa L.)

Molybdenum cofactor (Moco) is essential for nitrate reductase (NR), xanthine dehydrogenase (XDH), and aldehyde oxidase to perform their catalytic functions in plants. Moco biosynthesis is a complex process involving many genes. Little is known about the genetics and molecular aspects of Moco biosynthesis in plants and other eukaryotes. In rice, we previously isolated a Moco mutant C25 with a mutation in the CNX2 gene from a mutagenized indica cultivar IR30 and characterized its biochemical properties. This mutant was crossed with a japonica cultivar, Norin 8, to investigate the linkage of cnx2 to restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) markers. Chlorate resistance was used to trace the cnx2 mutation because of its cosegregation with the loss of NR and XDH activities observed earlier. RFLP and CAPS analyses show the location of the cnx2 locus on the long arm of chromosome 4. It is mapped between RFLP markers C513 and C377 with a distance of 9.5 and 13.1 cM, respectively. It is also linked with CAPS marker RA0738 at a distance of 30.3 cM. Received: 25 June 2000 / Accepted: 31 August 2000     

Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales,Chlorophyceae)

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild‐type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type‐specific and nitrate reductase‐related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii.     

Effects of hydroxylamine and molybdate on the formation of nitrate reductase in the yeast Rhodotorula

NADPH-nitrate reductase (NR) and NADPH-cytochrome c reductase(CR) activities of Rhodotorula glulinis var. salinaria cellsgrown in nitrate medium supplied with hydroxylamine (0.2 mM)were respectively 1.6- and 3.1-fold higher than those of cellsgrown without hydroxylamine. NR formed in nitrate plus hydroxylaminemedium is almost totally in an inactive form which is reactivatedin vitro by K₃Fe(CN)₆. When molybdate (10^–7 M) was suppliedto this medium, total (active plus inactive) NR activity increasedfurther about twofold but CR activity somewhat decreased. Inordinary nitrate medium, such molybdate effects were small.Most of the CR derepressed (induced) excessively in the nitrateplus hydroxylamine medium had a molecular size similar to NRon the basis of Bio-Gel A 1.5 m chromatography. Some other propertiesof CR formed in this medium were the same as those of the CRmoiety of NR. Adding molybdate to the nitrate plus hydroxylamine medium aftergrowing the cells for 20 hr induced the development of NR activitywithout any increase in CR activity. This induction was completelyblocked by cycloheximide, puromycin and L-canavanine but notcompletely by 6-methylpurine. Ammonium repressed this inductionwith markedly decreasing CR activity. The roles of hydroxylamine and molybdate in the formation ofNR are discussed. (Received May 26, 1980; )     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.     

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.     

nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.