Nitrate Reductase Activity Is Required for Nitrate Uptake into Fungal but Not Plant Cells

The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport. It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein. In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate. Uptake assays using the tracer (13NO3−) showed that nitrate influx is negligible in cells of a nitrate reductase null mutant. In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate. The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed. The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport. It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein. In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate. Uptake assays using the tracer (13NO3−) showed that nitrate influx is negligible in cells of a nitrate reductase null mutant. In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate. The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed. Nitrate is a major source of nitrogen for most algae, bacteria, fungi, and higher plants, and it is the nutrient that most frequently limits their growth (reviewed in Refs. 1Crawford N.M. Glass A.D.M. Plant Sci. 1998; 3: 385-395Google Scholar, 2Daniel-Vedele F. Filleur S. Caboche M. Curr. Opin. Plant Biol. 1998; 1: 235-239Crossref PubMed Scopus (148) Google Scholar, 3Williams L. Miller A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 659-688Crossref PubMed Scopus (236) Google Scholar). The first step in the assimilation of nitrate is the influx of nitrate into cells, which is an active process, because it can occur against an electrochemical potential gradient (4Vidmar J.J. Zhuo D. Siddiqi M.Y. Schjoerring J.K. Touraine B. Glass A.D.M. Plant Physiol. 2000; 123: 307-318Crossref PubMed Scopus (199) Google Scholar) followed by the catalytic activities of nitrate reductase and nitrite reductase that sequentially produces nitrite and ammonium, the latter being converted to organic nitrogen for cellular growth. Although there is considerable biochemical, genetical, and molecular biological information about these systems and their regulation, it is still not clear whether nitrate reductase is required for nitrate transport activity or whether transport occurs quite independently and in the absence of nitrate reductase activity (Ref. 5Wray J.L. Kinghorn J.R. Molecular and Genetical Aspects of Nitrate Assimilation. Oxford University Press, Oxford, United Kingdom1989Google Scholar and references therein). Mutants impaired in nitrate reductase activity have been studied to answer this question. Plant mutants with low levels of nitrate reductase in barley possess considerable nitrate transport activity (6King B.J. Siddiqi M.Y. Glass A.D.M. Plant Physiol. 1992; 99: 1582-1589Crossref PubMed Scopus (54) Google Scholar, 7Siddiqi M.Y. King B.J. Glass A.D.M. Plant Physiol. 1992; 100: 644-650Crossref PubMed Scopus (50) Google Scholar), and nitrate reductase-defective mutants in tobacco accumulate high levels of nitrate indicative of functional uptake (8Scheible W.R. Lauerer M. Schulze E.D. Caboche M. Stitt M. Plant J. 1997; 11: 671-691Crossref Scopus (375) Google Scholar). However, these mutants still possess some nitrate reductase activity making the interpretation of the importance of an active nitrate reductase for nitrate uptake somewhat equivocal, as low nitrate reductase activity alone might be sufficient to allow substantial transport. Fungi are more amenable to an intensive genetical approach, but, nevertheless, there are conflicting reports as to whether or not nitrate reductase activity per se is required for the expression of nitrate transport in fungi. For instance, studies carried out with Aspergillus nidulans implied an obligatory requirement (9Brownlee A.G. Arst Jr., H.N. J. Bacteriol. 1983; 155: 1138-1146Crossref PubMed Google Scholar), whereas the results in the related ascomycetous fungus Neurospora crassa (10Schloemer R.H. Garrett R.H. J. Bacteriol. 1974; 118: 258-269Google Scholar) showed complete autonomy. Recent work (11Machin F. Medina B. Navarro F.J. Perez M.D. Veenhuis M. Tejera P. Lorenzo H. Lancha A. Siverio J.M. Yeast. 2004; 21: 265-276Crossref PubMed Scopus (24) Google Scholar) in the nitrate-assimilating yeast Hansenula polymorpha shows that nitrate transport levels correlates with transporter protein levels and not nitrate reductase levels, although they did not assay nitrate uptake in a nitrate reductase-defective mutant. Here we demonstrate that nitrate reductase activity is mandatory for nitrate accumulation in cells of the lower eukaryotes, the fungi. The evidence comes first from heterologous studies with the A. nidulans NrtA (and NrtB) nitrate transporter proteins in the non-assimilating yeast Pichia pastoris and second from null nitrate reductase mutants of N. crassa and other nitrate-assimilating fungi. In contrast, studies of nitrate reductase null mutants in higher eukaryotic plant cells of Arabidopsis thaliana show that nitrate transport has no such obligatory requirement for nitrate reductase activity. Yeast Strains—P. pastoris GS115, a histidine-requiring derivative of the wild-type strain, which is unable to assimilate nitrate, and recombinant strain AtNia2, which possesses Arabidopsis nitrate reductase activity (12Su W. Huber S.C. Crawford N.M. Plant Cell. 1996; 8: 519-527Crossref PubMed Scopus (97) Google Scholar, 13Su W. Mertens J.A. Kanamaru K. Campbell W.H. Crawford N.M. Plant Physiol. 1997; 115: 1135-1143Crossref PubMed Scopus (33) Google Scholar, 14Skipper L. Campbell W.H. Mertens J.A. Lowe D.J. J. Biol. Chem. 2001; 276: 26995-27002Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), were used. Strains were maintained on yeast extract peptone dextrose medium (YPD) as recommended by Invitrogen. Filamentous Fungal Strains—Aspergillus fumigatus strains used in this study were wild-type, cnx1 and cnx3 mutants, Aspergillus niger wild-type and niaD101, and Aspergillus oryzae wild-type and niaD100 (all this study), and N. crassa wild-type, nit-3 (RIP), and nit-10 (RIP). 1G. Marzluf, unpublished. Mutant nit-3 (RIP) was found to lack nitrate reductase activity and nit-10 (RIP) nitrate transport activity (this study). Strain nit-1 is defective in the synthesis of the molybdenum cofactor (15Kramer S. Hageman R.V. Rajagopalan K.V. Arch. Biochem. Biophys. 1984; 233: 821-829Crossref PubMed Scopus (31) Google Scholar) and lacks nitrate reductase activity (this study). Mutant Aspergillus strains lacking nitrate reductase were selected on the basis of resistance to chlorate toxicity (16Cove D.J. Heredity. 1976; 36: 191-203Crossref PubMed Scopus (173) Google Scholar). All strains were maintained on Aspergillus complete medium (17Clutterbuck A.J. King R.C. Handbook of Genetics. 1. Plenum Publishing Corp., New York1974: 447-510Google Scholar). Fungal Net Nitrate Transport and Nitrate Reductase Assays—Conidial suspensions from filamentous fungi were inoculated into 200 ml of minimal medium containing 5 mm urea as the sole nitrogen source in 1-liter Erlenmeyer flasks, which were incubated with orbital shaking at 250 rpm. For A. niger, A. oryzae, and N. crassa incubation was carried out at 25 °C for 16–20 h and for A. fumigatus and A. nidulans at 37 °C for 6.5–7.5 h. To induce the nitrate assimilation pathway, 10 mm sodium nitrate was added 5 h before harvesting cells grown at 25 °C and 100 min before harvesting the cells grown at 37 °C. Yeast cultures were grown in 150 ml of YPD in baffled 500-ml Erlenmeyer flasks shaking at 200 rpm at 30 °C overnight to reach an A600 of 1.2–1.5. Cells were collected by centrifugation for 5 min at 2200 × g at room temperature and were washed with Pichia yeast nitrogen base (Invitrogen) containing 10 mm proline as the nitrogen source. Cells were resuspended in 150 ml of the same medium in baffled 500-ml Erlenmeyer flasks, and 0.5% methanol was added to induce the expression of genes under the control of the aox1 (alcohol oxidase) promoter. Flasks were incubated at 30 °C with shaking at 200 rpm for a total of 20 h with a further 0.5% methanol added after a 12-h incubation. Filamentous fungi were harvested by filtration through a miracloth (CN Biosciences, Nottingham, UK), and yeast cells were harvested by centrifugation as described above. Following washing in nitrate-free medium, net nitrate transport assays were carried out in minimal medium containing an initial concentration of 500 μm nitrate at 30 °C for P. pastoris, A. niger, A. oryzae, and N. crassa or at 37 °C for A. fumigatus and A. nidulans by the method described for A. nidulans (9Brownlee A.G. Arst Jr., H.N. J. Bacteriol. 1983; 155: 1138-1146Crossref PubMed Google Scholar). Aliquots were taken from the uptake medium at the start of incubation and after 20 min, wherein cells were removed rapidly by filtration through Millex filters (Millipore, Molsheim, France). Results are expressed as the nanomoles of nitrate depleted from the medium/min/mg of dry weight. N. crassa net nitrate transport assays were repeated using the original method developed for Neurospora (10Schloemer R.H. Garrett R.H. J. Bacteriol. 1974; 118: 258-269Google Scholar). Nitrate reductase assays were carried out according to the method described by Garrett and Cove (18Garrett R.H. Cove D.J. Mol. Gen. Genet. 1976; 149: 179-186Crossref PubMed Scopus (44) Google Scholar), and the results are expressed as the formation of nmol of nitrite/min/mg of protein. Fungal Uptake Assays Using the Tracer (13NO3−)—The growth of A. nidulans strains and the assay of nitrate influx were performed as detailed in Unkles et al. (19Unkles S.E. Zhou D. Siddiqi M.Y. Kinghorn J.R. Glass A.D.M. EMBO J. 2001; 20: 6246-6255Crossref PubMed Scopus (66) Google Scholar). Tracer experiments were conducted at the University of British Columbia, Vancouver, British Columbia, Canada. The routine concentration range of 250 μm nitrate was used. Growth of P. pastoris is described above, and the assay of nitrate influx was as detailed in Unkles et al. (19Unkles S.E. Zhou D. Siddiqi M.Y. Kinghorn J.R. Glass A.D.M. EMBO J. 2001; 20: 6246-6255Crossref PubMed Scopus (66) Google Scholar). Values for influx are expressed as nmol of nitrate/mg of dry weight/h. Escherichia coli Strains, Plasmids, and Media—Standard procedures were used for propagation of plasmids as well as for subcloning and maintenance of plasmids within the E. coli strain DH5α. Fungal Molecular Methods—DNA was isolated using a Nucleon BACC2 Kit (Amersham Biosciences). Total RNA was isolated using an RNeasy plant mini kit (Qiagen, Crawley, UK). The conditions used during dot blot analysis were as described previously (20Unkles S.E. Heck I.S. Appleyard M.V.C.L. Kinghorn J.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). was by analysis as described before (20Unkles S.E. Heck I.S. Appleyard M.V.C.L. Kinghorn J.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Yeast were performed as recommended by with for of for in P. of A. nidulans and genes were by from total RNA of the A. nidulans wild-type strain grown on nitrate as the sole nitrogen source. and the of the into the and and the of the into the of the expression (Invitrogen) to plasmids and The complete of both genes was using and and were from the Molecular University of United were and both from the and from the S.E. Zhou D. Siddiqi M.Y. Kinghorn J.R. Glass A.D.M. EMBO J. 2001; 20: 6246-6255Crossref PubMed Scopus (66) Google Scholar for were to at the of of the was used to in by a of and filamentous fungi were grown as for assays above, harvesting filamentous fungi by filtration and yeast by and yeast cells were washed with and were used or were in nitrogen Yeast cells were resuspended in ml of mm mm mm 25 mm used at °C containing mm 100 μm and one ml of was and cells were by for a total of min in with a on Filamentous fungi of of the were in and the was in 10 ml of from yeast and filamentous fungi were first by the centrifugation of suspensions at × g for 10 min at °C to and cells followed by the centrifugation of the at × g for 30 min at °C. The was resuspended in 150 of and were at °C. were carried out using a protein assay kit were by for 5 min in the of and mm the were for min, on PubMed Scopus Google Scholar), and to H. J. Sci. S. A. PubMed Scopus Google Scholar). were by incubation overnight at °C in mm sodium 20 mm containing (Amersham Biosciences). NrtA was by an incubation of the blot with in containing 0.5% for h at room temperature a washing with containing with for h at room further washing with activity was using (Amersham and (Amersham Biosciences). Arabidopsis nitrate reductase null strain was by the NIA2 deletion strain J. Crawford N.M. Plant Cell. 3: PubMed Scopus Google Scholar) with a containing a insertion in NIA1 S. M. D. M. Plant Cell. 1999; 11: PubMed Scopus Google Scholar). that were homozygous for the NIA1 insertion and NIA2 deletion were and no detectable nitrate reductase mRNA or activity not Arabidopsis Growth were grown with ml of medium in and with mm for 10 was added to the cultures to a concentration of μm as described by et al. M. Crawford N.M. Plant Physiol. PubMed Scopus Google Scholar). Arabidopsis Nitrate in and were by the method as described by et al. J.M. 1: Scopus Google Scholar). To nitrate were into in of the was into a and ml of was added to the and with the for The were in an for 10 min at The was removed for nitrate Net Nitrate Transport in P. pastoris, a Nitrate and containing the high nitrate transporter genes of A. and under the control of the yeast were transformed into the P. pastoris GS115, a yeast strain which not assimilate nitrate or possess nitrate reductase activity (12Su W. Huber S.C. Crawford N.M. Plant Cell. 1996; 8: 519-527Crossref PubMed Scopus (97) Google Scholar, 13Su W. Mertens J.A. Kanamaru K. Campbell W.H. Crawford N.M. Plant Physiol. 1997; 115: 1135-1143Crossref PubMed Scopus (33) Google Scholar, 14Skipper L. Campbell W.H. Mertens J.A. Lowe D.J. J. Biol. Chem. 2001; 276: 26995-27002Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). were selected on the basis of and the from selected were to the of the transporter the or gene were with the expression of the genes being by and net nitrate transport activity was detectable nitrate transport was observed in representative of GS115, such as and with the or not expression was by the of or strains were found to be to chlorate of to strain impaired chlorate (and expression of A. nidulans NrtA in the yeast P. pastoris and with recombinant nitrate reductase strain and nitrate transport nitrate in a and were transformed into P. pastoris AtNia2, a recombinant strain that the A. thaliana gene encoding nitrate reductase activity (12Su W. Huber S.C. Crawford N.M. Plant Cell. 1996; 8: 519-527Crossref PubMed Scopus (97) Google Scholar, 13Su W. Mertens J.A. Kanamaru K. Campbell W.H. Crawford N.M. Plant Physiol. 1997; 115: 1135-1143Crossref PubMed Scopus (33) Google Scholar, 14Skipper L. Campbell W.H. Mertens J.A. Lowe D.J. J. Biol. Chem. 2001; 276: 26995-27002Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). or were selected on the basis of the from was and or expression was by expression of net nitrate transport by the or genes not was observed in of the yeast strain nitrate reductase such as and The that nitrate was taken by and was by chlorate toxicity both strains being the ability to chlorate Net Nitrate Transport in Nitrate Reductase Strains of there are conflicting results in the filamentous we studies to Nitrate reductase loss-of-function mutants were on the basis of resistance to chlorate as described previously (16Cove D.J. Heredity. 1976; 36: 191-203Crossref PubMed Scopus (173) Google Scholar) for A. A. niger, and A. were found by and growth to be nitrate reductase gene mutants or to be in one of several molybdenum cofactor genes required for nitrate reductase activity. of the representative strains were found to possess nitrate reductase and no detectable net nitrate uptake was observed in of these strains nitrate uptake in nitrate reductase defective filamentous and reductase nitrate in a detectable uptake from the results from Aspergillus uptake was observed in the N. crassa nit-3 (RIP) strain, which is a of the nitrate reductase gene and is of nitrate reductase activity these results were at with the original (10Schloemer R.H. Garrett R.H. J. Bacteriol. 1974; 118: 258-269Google Scholar), the transport assays were repeated using growth conditions and assay according to the original method used for N. no net nitrate uptake was observed in the nitrate reductase defective nit-3 (RIP) or in a molybdenum cofactor defective nit-1 in N. crassa (15Kramer S. Hageman R.V. Rajagopalan K.V. Arch. Biochem. Biophys. 1984; 233: 821-829Crossref PubMed Scopus (31) Google Scholar). the lack of net nitrate transport in the N. crassa nit-10 (RIP) that this protein is for nitrate and the of this to on nitrate that it is the nitrate in N. crassa It is that nit-10 (RIP) still retained nitrate reductase although nitrate failed to the was observed for transporter mutants of A. nidulans (19Unkles S.E. Zhou D. Siddiqi M.Y. Kinghorn J.R. Glass A.D.M. EMBO J. 2001; 20: 6246-6255Crossref PubMed Scopus (66) Google Scholar). work (19Unkles S.E. Zhou D. Siddiqi M.Y. Kinghorn J.R. Glass A.D.M. EMBO J. 2001; 20: 6246-6255Crossref PubMed Scopus (66) Google Scholar, S.E. K. Campbell Kinghorn J.R. Sci. S. Google Sci. S. A. PubMed Scopus Google Scholar) has that in A. the is in or mutants lacking nitrate reductase activity. in P. pastoris, the was observed in such as and not lacking nitrate reductase activity. blot analysis was carried out using to whether the NrtA protein was in from yeast or filamentous cells with and nitrate reductase activity. major of was observed in a representative transformed P. pastoris strain, a transformant of that lacks nitrate reductase and in and not was from the strain but was in the P. pastoris transformant However, the protein was not observed in the nitrate strain or a in transformant of AtNia2, which no detectable nitrate uptake not of from A. nidulans nitrate reductase-defective strains showed a of to the wild-type It is that that the is the NrtA protein from studies of an A. nidulans deletion this strain was grown under it this not Fungal Uptake Assays Using the Tracer the that in nitrate reductase null mutants, nitrate influx was but a but for observed net in net nitrate uptake nitrate influx was using the tracer (13NO3−) on a selected the method of net nitrate uptake by the of nitrate from the uptake the tracer method tracer within of the fungus after a with A. nidulans nitrate reductase activity no nitrite was in nitrate reductase assays of grown under whereas the wild-type extract nmol of and net nitrate influx was with with for The results in showed that the A. nidulans negligible (13NO3−) influx that were to the strain Nitrate in a Nitrate Reductase A. thaliana a of h that the net nitrate uptake of a nitrate reductase null as by nitrate was to the wild-type nitrate to a higher in the and of the nitrate reductase null with the wild-type levels, because of the lack of nitrate reductase activity by wild-type and nitrate reductase null of A. reductase in a accumulation in wild-type and nitrate reductase null mutants of A. thaliana upon to of reductase null reductase null in a study was to whether nitrate reductase is required for nitrate uptake activity in the cells of fungi and plants, because studies were equivocal, to strains some nitrate reductase activity. were to cells for both lower and higher eukaryotic of nitrate reductase activity and to study their studies were carried out using the non-nitrate-assimilating yeast P. pastoris, which is a lower Net nitrate transport activity was in cells transformed with the A. nidulans nitrate transporter gene in the and absence of a recombinant A. thaliana nitrate reductase gene Net nitrate transport was observed in transformant cells but not nitrate reductase-defective mutants were in several filamentous fungi. Nitrate reductase loss-of-function mutants were and net nitrate N. crassa for which mutants were to possess nitrate uptake levels (10Schloemer R.H. Garrett R.H. J. Bacteriol. 1974; 118: 258-269Google Scholar). results from experiments using the tracer (13NO3−) that there is a of nitrate accumulation in strains lacking nitrate reductase activity. It is clear that yeast and filamentous fungi to accumulate nitrate in the complete absence of nitrate In contrast, in the higher eukaryotic plant, A. thaliana, nitrate transport activity is of nitrate reductase activity as by the of transport and the accumulation of nitrate in a loss-of-function nitrate reductase a strain by of both nitrate reductase genes, NIA1 and of nitrate uptake is not because of a of transporter gene expression or a lack of in fungi. It has been previously that nitrate reductase activity was not required for the of A. nidulans nitrate transporter and the in nitrate reductase loss-of-function mutants was with wild-type levels because of the of the (19Unkles S.E. Zhou D. Siddiqi M.Y. Kinghorn J.R. Glass A.D.M. EMBO J. 2001; 20: 6246-6255Crossref PubMed Scopus (66) Google Scholar, S.E. K. Campbell Kinghorn J.R. Sci. S. Google Sci. S. A. PubMed Scopus Google Scholar). was in this study of the nitrate non-assimilating P. pastoris, in which by was by The was in strains lacking nitrate it from blot experiments that the of nitrate transport activity in nitrate reductase strains is not because of a to the transporter as NrtA protein expression was that the lack of nitrate reductase activity to be the for the lack of nitrate regulation, and nitrate reductase from the same is not required to nitrate transport. Nitrate transport in yeast be in the of a plant nitrate and this that there is no requirement for nitrate reductase per se to be for transport activity. results the is the basis for the fundamental in nitrate reductase for nitrate influx these two eukaryotic The first is that there is a or nitrate reductase and the transporter protein in but not in plant In this nitrate, as well as being a a in as a for in formation and in H. J. 2000; PubMed Google Scholar, L. Plant Sci. 8: Full Text Full Text PDF PubMed Scopus Google Scholar). For these have a to the for nitrate because plant nitrate reductase can for the in second is that nitrate reductase is required to a gradient for nitrate into nitrate is not removed by nitrate reductase to nitrite of nitrate influx this is the can nitrate against a gradient in the absence of nitrate reductase whereas fungi that a lower for plant nitrate concentration be M.Y. Glass A.D.M. Plant Scopus Google Scholar). The answer in the of nitrate. Although the lower are of nitrate the higher eukaryote of nitrate for within the of to nitrate plant cells accumulate to of to mm nitrate (4Vidmar J.J. Zhuo D. Siddiqi M.Y. Schjoerring J.K. Touraine B. Glass A.D.M. Plant Physiol. 2000; 123: 307-318Crossref PubMed Scopus (199) Google Scholar). In addition, higher most of the nitrate to the for The maintenance of a gradient to nitrate is because the plant possesses no high nitrate transporter with one gene observed in the of most fungi and the of several of these plant genes be in in the absence of nitrate the nitrate concentration at the of plant cells be maintained at a low concentration by into the or by to the In other such of nitrate in plant cells an gradient to nitrate in the absence of nitrate reductase activity. In cells, the nitrate concentration at the rapidly in the absence of nitrate reductase activity to a concentration which further transport. it is somewhat that no (13NO3−) accumulation is observed in the absence of nitrate reductase because some accumulation into the might be at the gradient of nitrate was and the was more into these Marzluf, for mutants in the nitrate assimilation of N. W. and D. for recombinant strains of P. the University of British University for of

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