-
[show abstract]
[hide abstract]
ABSTRACT: The Chlamydomonas reinhardtii Nar1;1 gene encodes a membrane protein involved in nitrite transport to the chloroplast and is apparently dispensable for nitrate/nitrite-dependent growth in some conditions (Rexach, Fernández & Galván, Plant Cell 12, 1441–1454, 2000). In the present study the question of when NAR1;1 is required and how it improves nitrate utilization for growth was investigated. Comparative studies were performed with strains Nar1;1+ and Nar1;1– in nitrate media under three experimental conditions. Under continuous light and high CO2 (5%), strains containing or lacking Nar1;1 showed a very similar nitrate-dependent growth. Under continuous light and low CO2 (air), Nar1;1 allowed a slightly higher growth in nitrate. However, under light/dark cycles and low CO2, the strain having Nar1;1 showed a higher nitrate-dependent growth and doubled the amount of protein and chlorophyll than the Nar1;1– strain. The activities for nitrate uptake, nitrite and ammonium excretions, nitrate and nitrite reductases, glutamine synthetase (GS), as well as the transcript levels of Nar1;1, Nrt2;1, Nar2, Nrt2;3 and GS1 and GS2 genes were determined under this third condition by trying to understand how NAR1;1 improves nitrate use efficiency. Nitrite and ammonium excretion, which was higher in the strain Nar1;1– than in Nar1;1+, results in an important deregulation in the gene expression of nitrate transporters and cytosolic GS1. These data lead to the proposal that nitrite transport to the chloroplast mediated by NAR1;1 allows a balanced expression of enzymes and transporters for nitrate assimilation, thus optimizing nitrate utilization under the natural condition of light/dark cycles and air.
Plant Cell and Environment 10/2004; 27(10):1321 - 1328. · 5.22 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nitrate transport is the key step controlling the amount of nitrate incorporated by the cells and subsequent of storage, reduction or export. Molecular, genetic and biochemical approaches to the study of eukaryotic nitrate/nitrite transporters allow an initial understanding of this step, which is much more complex and structured than previously suspected. At the plasma membrane level, two gene families, Nrt1 and Nrt2, account for high- and low-affinity nitrate transporters. Functionality of NRT1 from Arabidopsis and NRT2 proteins from Aspergillus and Chlamydomonas has been demonstrated. However, redundancy of these systems makes it difficult to assign particular physiological roles to each. Data on genes involved in the regulation of nitrate transport and reduction are still scarce. Information on nitrite transporters to the chloroplast is biased by the belief that in vivo nitrous acid diffuses freely to this organellum. The recent progress on these aspects is discussed in this review.
Cellular and Molecular Life Sciences CMLS 03/2001; 58(2):225-33. · 6.57 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The nitrate reductase activity from Chlamydomonas reinhardtii was not altered when extracts were incubated with yeast 14-3-3 proteins in the presence of Mg-ATP. However, the C. reinhardtii extracts contained 14-3-3 proteins capable of inhibiting the spinach nitrate reductase, raising the question of their physiological substrates. Two C. reinhardtii proteins of about 48 and 35 kDa were eluted from 14-3-3 affinity chromatography columns and bound to 14-3-3s in overlay assays. The 48-kDa protein corresponded to the cytosolic isoform of glutamine synthetase (GS1). The GSI was phosphorylated by a Ca2+-and calmodulin-dependent protein kinase partially purified from the alga. However, neither phosphorylation nor 14-3-3 binding seemed to change GS catalytic activity.
Planta 02/2001; 212(2):264-9. · 3.00 Impact Factor
-
Trends in Plant Science 12/2000; 5(11):463-4. · 11.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO(2) conditions.
The Plant Cell 09/2000; 12(8):1441-53. · 8.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effect of nitrate on gamete differentiation as well as on the expression of genes involved in gametogenesis, nitrogen scavenging, and nitrate assimilation has been analyzed in wild-type and mutant strains of Chlamydomonas reinhardtii. Nitrate prevented gamete formation from wild-type strains and caused a strong reduction in the number of zygotes recovered in genetic crosses between nitrate-assimilation-deficient mutants, thus suggesting that nitrate by itself is providing a negative regulatory signal for the sexual differentiation of the alga. Addition of nitrate at low concentrations to wild-type cells, after an initial period of nitrogen starvation, resulted in a drastic decrease in transcript levels of both nitrate-assimilation genes (NIA1 and NRT2;1) and genes induced after N-starvation (NCG2 and NCG4). This strong effect of nitrate was due to its assimilation products since it was not evident in nitrate-assimilation mutants. A slight negative effect of nitrate on NCG4 expression was only observed in the mutant. Nitrate by itself was also found to provide a negative signal for the expression of gamete-specific genes (GAS3 and GAS18) in mutants incapable of assimilating nitrate.
Planta 08/2000; 211(2):287-92. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We constructed mutant strains lacking the nitrite reductase (NR) gene in Chlamydomonas reinhardtii. Two types of NR mutants were obtained, which either have or lack the high-affinity nitrate transporter (Nrt2;1, Nrt2;2, and Nar2) genes. None of these mutants overexpressed nitrate assimilation gene transcripts nor NR activity in nitrogen-free medium, in contrast to NR mutants. This finding confirms the previous role proposed for NR on its own regulation (autoregulation) and on the other genes for nitrate assimilation in C. reinhardtii. In addition, the NR mutants were used to study nitrate transporters from nitrite excretion. At high CO(2), only strains carrying the above high-affinity nitrate transporter genes excreted stoichiometric amounts of nitrite from 100 microM nitrate in the medium. A double mutant, deficient in both the high-affinity nitrate transporter genes and NR, excreted nitrite at high CO(2) only when nitrate was present at mM concentrations. This suggests that there exists a low-affinity nitrate transporter that might correspond to the nitrate/nitrite transport system III. Moreover, under low CO(2) conditions, the double mutant excreted nitrite from nitrate at micromolar concentrations by a transporter with the properties of the nitrate/nitrite transport system IV.
Plant physiology 02/2000; 122(1):283-90. · 6.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two high affinity nitrite transporters have been identified in Chlamydomonas reinhardtii. They have been named system III and system IV and shown to be differentially regulated by nitrogen and carbon supply. System III was induced under high CO(2) and required a micromolar nitrate signal for optimal expression, was inhibited by ammonium, and was not affected by either chloride or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid. System IV was induced optimally under limiting CO(2) and did not require nitrate signal, was inhibited by chloride and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but was not affected by ammonium. Two transcripts that shared the expression pattern of systems III and IV activities were detected with an Nrt2;3 gene probe. In addition, a mutant defective in both the activity of system III and the expression of Nrt2;3 gene has been isolated. Genetic crosses and in vivo complementation studies indicate that this mutant is defective in a locus that is closely linked to the regulatory gene Nit2.
Journal of Biological Chemistry 10/1999; 274(39):27801-6. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3'-end of the high-affinity nitrate transporter (HANT) gene Nrt 2; 2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified. Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark.
Planta 11/1998; 206(2):259-65. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci.
MGG - Molecular and General Genetics 06/1998; 258(4):373-7.
-
[show abstract]
[hide abstract]
ABSTRACT: Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the corresponding Chlamydomonas 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, named Nrg1 and Nrg2. 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 (I4C-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 that Nrg1 and Nrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway in C. reinhardtii.
MGG - Molecular and General Genetics 07/1996; 251(4):461-71.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
MGG - Molecular and General Genetics 05/1996; 251(4):461-471.
-
[show abstract]
[hide abstract]
ABSTRACT: Nitrate transport mutants from Chlamydomonas reinhardtii and strains derived from them upon transformation with plasmids containing the C. reinhardtii nar2/Nrt2;1 or nar2/Nrt2;2 genes have been used to study nitrate and nitrite transport systems. Mutants lacking nitrate assimilation clustered genes showed a high affinity nitrite transporter activity (system 3), which was subject to ammonium inhibition and appeared to be independent of a functional nar2 gene. Transformants carrying nar2/Nrt2;2 recovered a high affinity nitrate transporter activity (system 2) and showed nitrite transport activities with properties similar to those in nonrecovered high affinity nitrate transporter activity (system 1) together with a considerably enhanced nitrite transport activity. Nitrite transport mediated by system 1 was very sensitive to inhibition by nitrate at microM concentrations. Results strongly suggest that three nitrate assimilation related high affinity transport systems operate in C. reinhardtii: one specific for nitrite, a second one encoded by nar2/Nrt2;2 specific for nitrate, and another one encoded by nar2/Nrt2;1, which is bispecific for these two anions.
Journal of Biological Chemistry 02/1996; 271(4):2088-92. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The nitrite reductase (NiR) gene (nirA) has been isolated and sequenced from the filamentous, thermophilic non-N2-fixing cyanobacterium Phormidium laminosum. Putative promoter-like and Shine-Dalgarno sequences appear at the 5' end of the 1533 bp long nir-coding region. The deduced amino acid sequence of NiR from P. laminosum corresponds to a 56 kDa polypeptide, a size identical to the molecular mass previously determined for the pure enzyme, and shows a high identity with amino acid sequences from ferredoxin-dependent NiR. This cyanobacterial NiR gene has been efficiently expressed in Escherichia coli DH5 alpha from the E. coli lac promoter and probably from the P. laminosum NiR promoter.
Plant Molecular Biology 04/1995; 27(5):1037-42. · 4.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Chlamydomonas reinhardtii nar-2, nar-3, and nar-4 genes, which are within a nitrate-regulated gene cluster containing the nitrate reductase structural gene nit-1, have been related to nitrate transport. Mutant strains defective in nitrate transport and having an active nitrate reductase have been genetically constructed. Their nitrate non-utilizing phenotype has been directly complemented by transformation using the pCO-5 plasmid which carries the nar-2, nar-3, and nar-4 clustered genes. Integration of pCO-5 DNA in the genome of nitrate transport mutants resulted in the expression of these nar transcripts and the recovery of a high affinity nitrate transport activity. Complementation of the nitrate non-utilizing phenotype of the constructed strains was also achieved by co-transformation with plasmids containing nar-2 and nar-3 genes or nar-2 and nar-4, but not with single plasmids containing each individual gene. In addition, DNA sequences of a practically complete cDNA of nar-3 and a partial one of nar-4 have been generated and the deduced amino acid sequences showed a very significant identity with that of the nitrate transporter gene (crnA) from Aspergillus nidulans. These data strongly support the hypothesis that the nitrate transport system in C. reinhardtii contains at least two protein components encoded by the nar-2 and nar-3 genes. The nar-4 gene would produce a protein with a high identity to that of nar-3.
The Plant Journal 04/1994; 5(3):407-19. · 6.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The mRNA accumulation pattern of the Chlamydomonas reinhardtii nitrate assimilation-related gene cluster has been elucidated. In ammonium-grown wild-type cells, nit-1 (nitrate reductase, NR), nar-1, nar-2 and nar-3 (nitrate transporter) genes showed very similar kinetics of expression when transferred to nitrate medium. Transcripts of all these genes accumulated transiently in ammonium-grown wild-type cells after a one-hour incubation in nitrogen-free medium, and practically disappeared at about 2 hours. Mutant strains lacking functional nitrate reductase showed similar accumulation kinetics of these transcripts during both nitrate induction and derepression in nitrogen-free media. In contrast to the other nar transcripts, that nar-4, a gene sharing similar sequences with nar-3, accumulated in small amounts in wild-type cells, and only increased after a long nitrate induction period. Nitrate and light showed a strong positive effect on the accumulation of nit-1 gene transcripts. Acetate as a carbon source allowed accumulation of nit-1 mRNA in the dark, indicating the existence of interactions between light and carbon metabolism in nit-1 gene expression. Our data strongly suggest that NR negatively autoregulates its own expression and that of nar genes.
Plant Molecular Biology 02/1994; 24(1):185-94. · 4.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Three overlapping clones covering a Chlamydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1. These new loci produced transcripts of 2.8, 2.2, 1.8 and 1.7 kb in nitrate-induced wild-type cells that, like the 3.4 kb transcript of nit-1, were undetectable in cells grown in ammonium. In addition, in a mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express the nit-1 gene transcript, accumulation of the four other transcripts was also blocked. They have been named nar (nitrate assimilation related) genes. The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1. The nar-3 and nar-4 loci are transcribed divergently from nit-1. DNA and RNA sequences from both nar-3 and nar-4 cross-hybridized with each other indicating that they share similar sequences. Four nitrate assimilation-deficient mutants (C2, D2, F6 and G1) were characterized. These mutants lack nar transcripts and have major deletions and/or rearrangements in the nar gene cluster. In contrast to other nitrate reductase-deficient mutants and to wild type, deletion mutants and the regulatory mutant nit-2 were incapable of accumulating intracellular nitrate. Two of the mutants in which expression of all the nar loci did not occur, C2 and D2, grew in nitrite medium and showed wild-type levels of both nitrite uptake and nitrite reductase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
MGG - Molecular and General Genetics 10/1993; 240(3):387-94.
-
[show abstract]
[hide abstract]
ABSTRACT: A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.
Biochimica et Biophysica Acta 01/1993; 1160(3):269-74. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture. Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.
FEBS Letters 08/1992; 307(2):162-3. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Expression of nitrite uptake and nitrite reductase activities has been studied in Chlamydomonas reinhardtii under different nutritional conditions. Both activities were expressed at a low level in derepressed cells (with no nitrogen source) and at a high level in induced cells (with nitrate or nitrite). Nitrate was required for both activities to be maximally expressed. Ammonium-grown cells did not show nitrite uptake capability and had a basal nitrite reductase activity. Nitrite uptake but not nitrite reductase levels decreased very significantly in nitrate-induced cells subject to cycloheximide treatment, which suggests that protein(s) involved in the uptake are under a rapid turnover. Nitrite uptake expression was strongly inhibited by the presence of the glutamine synthetase inhibitor L-methionine-D,L-sulfoximine under either derepression or induction conditions, whereas that of nitrite reductase was not affected under the same conditions. Our results indicate that nitrite uptake expression is regulated primarily by ammonium, and that of nitrite reductase by both ammonium and ammonium derivative(s).
Biochimica et Biophysica Acta 06/1991; 1074(1):6-11. · 4.66 Impact Factor
