FEMS Yeast Research 4 (2003) 149^155
www.fems-microbiology.org
The role of nitrate reductase in the regulation of the nitrate
assimilation pathway in the yeast
Hansenula polymorpha
Francisco J. Navarro a , Germa¤n Perdomo a;1 , Paula Tejera a , Braulio Medina a ,
Fe¤lix Mach|¤n a;2 , Rosa M a . Guille¤n a;3 , Ana Lancha b , Jose¤ M. Siverio a;
a
b
Departamento de Bioqu|¤mica y Biolog|¤a Molecular, Grupo del Metabolismo del Nitro¤geno, Universidad de La Laguna, E-38206 La Laguna,
Tenerife, Canarias, Spain
Departamento de Microbiolog|¤a y Biolog|¤a Celular, Grupo del Metabolismo del Nitro¤geno, Universidad de La Laguna, E-38206 La Laguna,
Tenerife, Canarias, Spain
Received 19 March 2003; received in revised form 17 June 2003; accepted 14 July 2003
First published online 26 August 2003
Abstract
The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula
polymorpha. Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the
transcriptional regulation of nitrate-assimilatory gene expression was examined. The strain bearing YNR1 (nitrate reductase gene) under
the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen
sources. In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar
to that in cells induced by nitrate alone. This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of
H. polymorpha nitrate assimilation genes. To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1,
or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used.
Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain. Moreover, in strains
lacking the high-affinity nitrate transporter and NR this deregulation disappeared. These facts discard the role of NR protein in the
transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part
of the nitrate-signalling pathway.
6 2003 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
Keywords : Nitrate reductase; High-a⁄nity nitrate transport; Nitrate regulation ; Rapamycin ; Yeast; Hansenula polymorpha
1. Introduction
The yeast Hansenula polymorpha is able to use nitrate as
* Corresponding author. Tel. : +34 (922) 318 406;
Fax : +34 (922) 318 311.
E-mail address : [email protected] (J.M. Siverio).
1
Present address: Division of Endocrinology and Metabolism,
Department of Medicine, University of Pittsburgh, 200 Lothrop St.,
Pittsburgh, PA 15231, USA.
2
Present address: Cell Cycle Group, MRC Clinical Sciences Centre,
Imperial College London, Hammersmith Hospital Campus, Du Cane
Road, London W12 0NH, UK.
3
Present address: Universidad Nacional de Asuncio¤n, Facultad de
Ciencias Qu|¤micas, Asuncio¤n, Paraguay.
single nitrogen source. Nitrate is transported into the cell
by the high-a⁄nity nitrate transporter Ynt1 [1]. However,
nitrate and nitrite transport independent of Ynt1 has also
been observed [2]. H. polymorpha responds to the presence
of nitrate in the medium by expressing the nitrate-assimilatory genes YNT1 (high-a⁄nity nitrate transporter),
YNR1 (nitrate reductase) and YNI1 (nitrite reductase)
[3,4]. Two highly similar Zn(II)2 Cys6 transcriptional factors encoded by the genes YNA1 and YNA2 have been
found to be indispensable for nitrate induction in H. polymorpha. Yna1 and Yna2 present high similarity with their
counterparts NirA and NIT4 in the ¢lamentous fungi Aspergillus nidulans and Neurospora crassa [3,4]. A very similar mechanism of nitrate induction has been depicted in
A. nidulans and in N. crassa. In A. nidulans two positive
1567-1356 / 03 / $22.00 6 2003 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
doi:10.1016/S1567-1356(03)00163-6
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factors are involved in nitrate induction; NirA, a nitratespeci¢c pathway transcription factor and the GATA factor-type AreA involved in nitrogen catabolite repression
[5]. Intracellular nitrate also seems to play an important
role in nitrate induction, since in vivo the dependence of
NirA DNA binding on intracellular nitrate has been established [6]. The necessity of intracellular nitrate to trigger nitrate induction has also been shown in Chlamydomonas reinhardtii, in such a way that nitrate transporters play
a key role in nitrate induction [7].
Nitrate reductase (NR) appears to be a key point in the
regulation of nitrate assimilation in most organisms. However, di¡erent organisms have evolved distinct mechanisms
to control NR activity. In plants, NR levels are regulated
transcriptionally as a response to endogenous and environmental factors such as nitrogen source, light, hormones, etc. In addition, posttranslational regulation has
been observed as a response to light^dark cycles, photosynthetic activity and CO2 levels [8]. This regulation involves protein phosphorylation and 14-3-3 proteins binding to the phosphorylated NR site [8,9]. In C. reinhardtii
reversible inactivation by reduced nitrogen sources has
also been found [10]. With regard to yeasts, there are
species able to use nitrate and ammonium simultaneously
such as Hansenula anomala (Siverio and Garcia-Lugo, unpublished results), Candida nitratophila and Candida utilis
[11], suggesting that ammonium neither severely represses
nitrate-assimilatory genes nor inactivates the components
of the pathway. On the other hand, in yeast species such
as Sporobolomyces roseus and Rhodotorula glutinis [11] and
H. polymorpha (Siverio and Mach|¤n, unpublished results)
ammonium or glutamine inactivates nitrate consumption.
In these yeasts it seems that nitrogen catabolite repression
does not account for the decrease in nitrate consumption
observed after ammonium or glutamine addition to nitrate-grown cells. We studied here how reduced nitrogen
sources a¡ect NR activity and protein stability in H. polymorpha, since NR along with nitrate transporters could be
responsible for this fact.
NR has also been involved in the transcriptional regulation of nitrate assimilation genes. A fact largely observed
in ¢lamentous fungi and algae is that mutants lacking NR
activity present a deregulation of nitrate-assimilatory gene
expression, in such a way that in a nitrate-free medium
nitrate-assimilatory genes are expressed. This behavior has
been claimed by several authors to be a phenomenon of
autogenous regulation [12^16], that is, the regulation of
the nitrate-assimilatory genes depends on the NR protein.
The mechanism proposed is that in the absence of nitrate,
NR would interact with a transcriptional factor involved
in nitrate induction (NIRA, NIT4, Yna1, Yna2) [17^19],
thereby inactivating its positive regulatory e¡ect [12,15,16].
In the presence of nitrate the complex would dissociate,
allowing the transcriptional factor to interact with the nitrate-assimilatory genes. In the absence of NR protein, the
transcriptional factor involved in nitrate induction would
interact with nitrate-assimilatory genes allowing their expression even in the absence of nitrate [12,15,16]. However, no direct evidence of the involvement of NR in
this mechanism has been shown. An alternative explanation would be that nitrate traces, in an apparently nitratefree medium, would accumulate intracellularly due to the
lack of NR, resulting in nitrate-assimilatory gene induction (gratuitous induction) [15]. In high-a⁄nity nitrate
transporter C. reinhardtii mutants, in agreement with the
latter hypothesis, the deregulation of the nitrate-assimilatory genes in an NR mutant is abolished. Furthermore,
preliminary evidence, consistent with the gratuitous induction, was obtained by an H. polymorpha strain lacking the
NR gene (vynr1 : :URA3) and expressing tobacco NR
under the YNR1 gene promoter [20].
In the frame of our studies on the regulation of the
nitrate assimilation pathway, in this work we focus our
attention on the role of NR. We found that ammonium
or other reduced nitrogen sources have no, or only slight,
posttranscriptional e¡ects on NR activity. Concerning the
role of the endogenous NR on the transcriptional regulation of the nitrate-assimilatory genes, tobacco NR abolished the deregulation e¡ect observed in the NR-minus
mutant. This shows that endogenous NR by itself has
no direct involvement in the nitrate induction system.
We also found that in a double mutant strain, lacking
the NR gene and the high-a⁄nity nitrate transporter, no
deregulation occurred.
2. Materials and methods
2.1. Growth conditions, yeast strains and plasmid constructs
Yeasts were grown at 37‡C with shaking in liquid medium containing 0.17% w/v yeast nitrogen base without
ammonium sulfate and amino acids (Difco), 2% w/v glucose and 5 mM ammonium chloride as nitrogen source,
unless otherwise stated. Yeast transformation was done
according to [21].
All strains used in this work were derivatives of H.
polymorpha NCYC495 leu2 ura3 (Table 1). The strain
GP100 was constructed by targeted integration of
pGP16, linearized at StuI site to the MOX promoter.
The plasmid pGP16 was constructed to express YNR1
under the control of the MOX promoter. A 2735-bp
blunt-ended EcoRI^KpnI DNA fragment containing the
YNR1-coding region was cloned into the SmaI site of
pET1 [23]. The FN12003 strain was created by targeted
integration of plasmid pGP1, linearized at the BstEII site,
Ł vila,
to the leu2 locus of a vynr1 : :URA3 leu2 strain (J. A
1995). pGP1 carries a sequence of 938 bp from the 5P noncoding region of the YNR1 gene plus 21 bp of the coding
region upstream lacZ [22]. Cotransformation of strain
vynr1: :URA3 leu2 with pGP1 and pGPC20 plasmids, linearized at the BstEII and BclI sites, respectively, yielded
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the FN22003 strain. pGPC20 contains Nia2 cDNA
£anked by the 5P and 3P non-coding regions of the
YNR1 gene [20]. pGP1 and pGPC20 were integrated at
the leu2 locus and the 3P non-coding region of the
YNR1 gene, respectively. FN32003 was obtained by integration of BstEII-linearized pGP1 at the leu2 locus of a
vynt1: :vura3 leu2 strain and simultaneously by disruption
of the chromosomal gene YNR1 following the method
described in [24]. The strain vynt1 : :vura3 leu2 was obtained from vynt1: :URA3 leu2 [1] after disruption of
the chromosomal URA3 gene with a polymerase chain
reaction (PCR) product of vura3 bearing an internal deletion.
To produce the NR protein region (492^718 residues) in
Escherichia coli, the 681-bp XhoI^PstI DNA fragment
from the YNR1-coding region [25] was cloned into the
XhoI^PstI sites of the pRSET A vector (Invitrogen, Carlsbad, CA, USA), thus obtaining the plasmid pGP2.
2.2. Polyacrylamide gel electrophoresis (PAGE), Western
blot and enzyme activities
Crude extracts were obtained in the presence of the
protease inhibitor cocktail Complete Mini (Roche, Rotkreuz, Switzerland) as described [26]. Proteins subjected to
sodium dodecyl sulfate (SDS)^PAGE were transferred to
polyvinylidene di£uoride (PVDF) membranes (Amersham,
Sunnyvale, CA, USA) according to the manufacturer’s
instructions. Immunodetection of NR was done with a
chemiluminescence system (Amersham). NR and L-galactosidase activities were measured according to [22,25], respectively. NR activity is expressed in nmol of nitrite
min31 (mg protein)31 .
151
Fig. 1. Expression of YNR1 under control of the MOX promoter in the
presence of ammonium or nitrate. H. polymorpha strain bearing the
YNR1-coding region fused to the MOX gene promoter was grown in
ammonium-containing medium. Cells were transferred to the derepression medium (0.17% w/v yeast nitrogen base without ammonium sulfate
and amino acids, 1% v/v glycerol, 5 mM ammonium) and incubated for
2 h. Afterwards, cells were transferred to the induction medium (0.17%
w/v yeast nitrogen base without ammonium sulfate and amino acids,
0.5% v/v methanol, 5 mM nitrate or ammonium), with addition of
methanol every 12 h of incubation. NR activity and protein were followed after methanol addition in medium with nitrate (A) or ammonium (B). To determine NR protein levels, 20 Wg of protein were analyzed by Western blot. Two independent experiments were done without
signi¢cant deviation.
3. Results
3.1. E¡ect of reduced nitrogen sources on NR protein and
activity in cells expressing YNR1 under the control of
the MOX promoter
2.3. Preparation and characterization of anti-NR antiserum
NR antigen was obtained in the E. coli BL21(DE3)pLysS strain (Invitrogen) transformed with the pGP2 plasmid. Expression was achieved as described by Invitrogen.
The antiserum was prepared in rabbit [27]. In Western
blot, the antiserum 1:3000 diluted recognized a protein
of 104 kDa in cells from nitrate medium, but not from
ammonium medium, nor in the vynr1: :URA3 mutant incubated with nitrate (data not shown).
To examine the importance of reduced nitrogen sources
for NR activity, YNR1 was expressed under the control of
the H. polymorpha MOX gene promoter to bypass the
transcriptional regulation of YNR1 by nitrogen sources.
MOX is derepressed by glycerol, induced by methanol
and repressed by glucose [28,29]. In a strain, bearing the
fusion MOX-YNR1, YNR1 was induced by methanol with
ammonium or nitrate as nitrogen source. NR activity appeared as much in ammonium as in nitrate medium. In
Table 1
Yeast strains
Strains
a
NCYC495 (WT)
GP100
YNRgal
FN12003
FN22003
FN32003
Genotype
Source
leu2 ura3 LEU2 URA3
vynr1: :URA3 PMOX : :pGP16(PMOX -YNR1-TAMO1 HpLEU2)
leu2: :pGP1(PYNR1 -lacZ HpLEU2)
vynr1: :URA3 leu2: :pGP1(PYNR1 -lacZ HpLEU2)
vynr1: :URA3: :pGPC20(PYNR1 -Nia2-TYNR1 ) leu2: : pGP1 (PYNR1 -lacZ HpLEU2)
vynt1: : vura3 vynr1 : :URA3 leu2: : pGP1 (PYNR1 -lacZ HpLEU2)
NMG
this work
[22]
this work
this work
this work
a
This strain was obtained at the Nitrogen Metabolism Group (NMG) by transforming the NCYC495 leu2, ura3 with plasmids linearized at the
HpLEU2 and HpURA3 genes.
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between activity and protein was observed. While with
ammonium the activity decreased with respect to the protein, with nitrate it increased. The decrease of the activity
in ammonium medium could be due to methanol levels in
the medium after 2 days of growth. In spite of the fact that
methanol was added to the medium every 12 h, in ammonium plus methanol the cells grew faster than in nitrate
plus methanol, hence methanol was exhausted faster with
ammonium than in nitrate. The synthesis of NR prosthetic
groups could be hindered in the absence of carbon sources, leading to a pool of inactive NR.
3.2. E¡ect of nitrogen source on NR activity and protein in
the wild-type strain
Despite the results above, to avoid possible side e¡ects
of methanol, we further studied the e¡ect of reduced nitrogen sources on NR levels in the wild-type strain. Di¡erent
nitrogen sources were added to the cells, after being incubated in nitrate medium for 2 h to induce NR. As shown
in Fig. 2A and B, no important di¡erences in the amount
of NR protein or in the activity were observed in response
to the presence of di¡erent nitrogen sources. Furthermore,
a good correlation between the amount of NR protein and
activity was observed in all cases studied. The slight e¡ect
of preferred nitrogen sources such as ammonium, glutamine and asparagine on NR activity and protein is remarkable. NR activity in nitrite slightly increased, since
nitrite is a YNR1 inducer [4].
3.3. In the presence of rapamycin NR is active with nitrate
plus glutamine
Fig. 2. E¡ect of di¡erent nitrogen sources on the levels of NR and
NR :NADH activity in a wild-type strain. Wild-type cells were grown in
ammonium medium, washed and transferred to 5 mM nitrate for 2 h to
induce NR. Afterwards, cells were transferred to media containing 5 mM
ammonium (F), 1 mM nitrite (b), 1 mM proline (a), 1 mM asparagine
(O), 1 mM glutamine (E), or without nitrogen source (R). In A, 20 Wg
of protein were analyzed by Western blot; B shows NR:NADH activity. In the experiments shown 100% corresponds to 42 mU (mg
protein)31 . Two independent experiments were carried out without signi¢cant deviation.
addition, the levels of NR protein were correlated with the
activity, being both protein and activity two to three times
higher with ammonium than with nitrate (Fig. 1). However, in samples corresponding to 60^72 h, less correlation
Rapamycin is a lipophilic macrolide with both antifungal and immunosuppressive activities. It forms a complex
with a small 12-kDa peptidyl-prolyl isomerase, FKBP12
[30]. This complex is a potent inhibitor of Tor1 and Tor2
(target of rapamycin) activity. With preferred nitrogen
sources, such as ammonium or glutamine, Tor1 and
Tor2 are active; as a result, in Saccharomyces cerevisiae
the genes subjected to nitrogen catabolite regulation are
repressed. On the contrary, with non-preferred nitrogen
sources or in the presence of preferred nitrogen sources
plus rapamycin, the repression undergone by nitrogen catabolite repression-sensitive genes is released [31^34]. The
target of the Tor signal transduction pathway, concerning
nitrogen catabolite repression, is the positive GATA transcriptional factor Gln3 [35,36]. Rapamycin seems to be an
excellent tool to study the posttranslational e¡ect of reduced nitrogen sources on the activity of enzymes encoded
by genes subjected to nitrogen catabolite repression. We
focussed here our attention on the e¡ect of rapamycin on
NR levels in the presence of reduced nitrogen sources.
Firstly, it was established that in a strain expressing lacZ
under the YNR1 promoter, nitrate induction of L-galactosidase was not a¡ected by reduced nitrogen sources in the
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153
Fig. 3. E¡ect of rapamycin on NR activity in glutamine medium plus nitrate. Wild-type ammonium-grown cells were thoroughly washed and incubated
for 1.5 h in 2.5 mM sodium nitrate, 5 mM glutamine plus 2.5 mM nitrate, and 5 mM glutamine, as indicated in the ¢gure. Glutamine-containing media
with or without nitrate were supplemented with either 0.5 Wg ml31 rapamycin or with only drug vehicle (ethanol 90%, Tween-20 10%). Rapamycin was
prepared in drug vehicle at 1 mg ml31 . After incubation in these media NR activity was determined. Means Y standard deviations of two independent
experiments are represented. 100% NR:NADH activity corresponds to 32 mU (mg protein)31 .
presence of rapamycin (data not shown). Therefore, rapamycin abolishes nitrogen catabolite repression in H. polymorpha in the same way as reported in S. cerevisiae [31].
This ¢nding was used as an alternative approach to assess
the e¡ect of reduced nitrogen sources on NR. Cells incubated in glutamine plus nitrate and glutamine plus nitrate
plus rapamycin (Fig. 3) revealed a minor e¡ect, if any, of
the reduced nitrogen sources on NR activity.
In summary, evidence from di¡erent experiments shows
that the stability and activity of NR is not a¡ected by
reduced nitrogen sources. This fact points out that NR
is not involved in the inactivation of nitrate consumption
observed after glutamine addition. Therefore, the high-af¢nity nitrate transporter is probably the main target in the
elimination of the £ux through the nitrate pathway in
response to reduced nitrogen sources.
type strain would be in disagreement with this hypothesis.
As shown in Fig. 4, in a medium lacking nitrogen sources,
the strain bearing tobacco NR (FN22003), unlike the NR
gene disruptant strain (FN12003), behaved similarly to the
3.4. Expression of tobacco NR or disruption of the
high-a⁄nity nitrate transporter (YNT1) abolishes
nitrate-assimilatory gene deregulation present in
NR mutants
To challenge the hypothesis attributing to NR a regulatory role in the nitrate-assimilatory gene expression, a
plant NR was expressed under the YNR1 gene promoter.
It was assumed that a heterologous plant NR is incapable
of maintaining the putative interaction with the positive
transcriptional factors involved in nitrate induction.
Under this assumption, the strain expressing tobacco
NR would behave in agreement with the autogenous regulation hypothesis, that is, deregulation in nitrate-free medium [12,15,16]. On the contrary, a response as in a wild-
Fig. 4. Expression of tobacco NR abolishes nitrate assimilation gene deregulation. Ammonium-grown cells were washed, resuspended and incubated for 7 h in a nitrogen-free medium. At the indicated times, samples
from the cultures were collected to determine L-galactosidase activity.
100% Activity corresponds to 164.5 nmol o-nitrophenol min31 (mg
protein)31 . The experiment was repeated three times without signi¢cant
di¡erences ; results from only one experiment are shown. YNRgal :
YNR1-lacZ ; FN12003 : ynr1 YNR1-lacZ ; FN22003 : ynr1 YNR1-lacZ
YNR1-Nia2 ; FN32003 : ynr1 YNR1-lacZ ynt1.
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wild-type strain. These results demonstrate that the constitutive expression of the nitrate-assimilatory genes observed in the NR-minus strain is due to a lack of NR
activity and the concomitant intracellular nitrate accumulation. The expression of a heterologous NR restored the
wild-type behavior of a strain lacking the endogenous NR,
thus discarding the role of fungal NR as a part of the
transcriptional machinery acting in response to nitrate.
To prove that nitrate traces present in a medium ‘free of
nitrogen sources’ were responsible for the induction of
nitrate-assimilatory genes, a strain vynr1: :URA3 vynt1
: :URA3 expressing lacZ under the YNR1 promoter was
used (FN32003). In this strain, after 7 h in a nitrogen-free
medium, no L-galactosidase activity was observed. This
supports the hypothesis that nitrate traces accumulating
in an NR mutant are responsible for nitrate-assimilatory
gene deregulation, since in a mutant lacking the higha⁄nity nitrate transporter, nitrate traces cannot be transported into the cells. However, nitrate in the mM range
induced L-galactosidase activity in the double mutant
(ynt1 ynr1), due to the fact that nitrate enters the cells
through the low-a⁄nity nitrate transporter (data not
shown). These results are in agreement with those shown
in A. nidulans, where in vivo intracellular nitrate and AreA
were indispensable for NirA DNA binding [6]. Moreover,
in C. reinhardtii it was shown that nitrate signalling to the
NR promoter directly depends on the activity of nitrate
transporters [7].
4. Discussion
At short term, reduced nitrogen sources decrease consumption of extracellular nitrate in H. polymorpha. Nitrogen catabolite repression does not account for this fact
and therefore some of the components of the nitrate assimilation pathway must undergo some posttranscriptional
inactivation. The e¡ect of reduced nitrogen sources on NR
activity and protein was evaluated following three approaches: (1) expressing YNR1 under the MOX promoter
which is not subjected to nitrogen catabolite repression,
(2) studying a wild-type strain and (3) using rapamycin
which abolishes nitrogen catabolite repression. These three
di¡erent approaches were carried out to discard possible
side e¡ects of methanol or rapamycin on NR. The data
clearly show that NR is active in the presence of reduced
nitrogen sources such as ammonium or glutamine. However, posttranslational modi¢cations of NR without e¡ect
on NR activity cannot be discarded. Tobacco NR expressed under the control of the CaMV 35S promoter
was also active in presence of ammonium [37]. On the
contrary, in Chlamydomonas, the constitutive expression
of the NR gene in the presence of ammonium led to
an inactive enzyme [10]. In conclusion, the lack of NR
posttranscriptional regulation points out that nitrate
transporter(s) could be responsible for the glutamine or
ammonium inactivation of nitrate consumption in H. polymorpha.
The availability of vectors to express lacZ under di¡erent promoter sequences [22] as well as di¡erent expression
vectors [20] has contributed to increase the versatility of
H. polymorpha as a model organism to study nitrate assimilation [18]. As a result we were able to study the involvement of NR and the high-a⁄nity nitrate transporter
in the nitrate-assimilatory gene regulation. A matter
largely disputed has been the autoregulatory role attributed to NR. The expression of tobacco NR in a strain
with disrupted YNR1 gene revealed that tobacco NR restores all the functions of that endogenous NR, prompting
exogenous nitrate to fully induce L-galactosidase activity
(Fig. 4). It is concluded that NR activity and not the
protein itself is the important factor that abolishes nitrate-assimilatory gene deregulation, exhibited by strains
lacking NR activity.
Concerning the high-a⁄nity nitrate transporter, experiments presented in Fig. 4 reveal that in an NR-de¢cient
strain, in agreement with the gratuitous induction hypothesis [7,15], nitrate traces have to be transported into the
cells to induce nitrate-assimilatory genes. This ¢nding is in
agreement with results in A. nidulans and C. reinhardtii
[6,7]. In contrast, in an A. nidulans strain lacking both
high-a⁄nity nitrate transporters, evidence suggesting
extracellular sensing of nitrate has also been reported.
This mutant did not show either nitrate transport or nitrate growth, but nitrate-assimilatory genes were induced
[38].
In summary, here we report evidence on the minor role
of NR in the regulation of the nitrate assimilation pathway. It seems that the high-a⁄nity nitrate transporter has
a more relevant role than NR in the posttranscriptional
adaptation of nitrate £ux to the nitrogen demands. The
mechanisms involved in the posttranslational regulation of
the high-a⁄nity nitrate transporter constitute a challenge
for researchers in this ¢eld. Moreover, our results discard
the autoregulatory role of fungal NR and give sound evidence of the involvement of nitrate transporters in nitrate
sensing.
Acknowledgements
We thank J. Cregg (Claremont, CA, USA) for pET1
plasmid and advice on YNR1 expression under the control
of the MOX promoter, E. Ferna¤ndez (Co¤rdoba) and Claudio Scazzocchio (Paris) for priming experiments using heterologous NR. Grants from the Ministerio de Ciencia y
Tecnolog|¤a (BMC2001.3719) and Gobierno de Canarias
(PI2001/050) to J.M.S. supported this work. F.J.N. and
P.T. were recipient of predoctoral fellowships from the
Ministerio de Educacio¤n, Cultura y Deporte (Spain),
B.M. from the Gobierno de Canarias and R.M.G. from
Universidad Nacional de Asuncio¤n, Paraguay.
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