(Received for publication, September 20, 1995; and in revised form, November 13, 1995)
From the
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 nontransformed cells. Transformants carrying nar2/Nrt2;1 recovered 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 µM 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.
Nitrate, the preferred nitrogen source in photosynthetic organisms, is assimilated by a highly regulated pathway whose first step is the entry of nitrate into the cells mediated by an active transport system(1, 2, 3) . In plant cells, it is believed that at least two transport systems are involved in nitrate uptake. One is a high affinity system that is nitrate inducible (4, 5, 6) and another one is a low affinity system that is constitutively expressed(7) . An intensive effort has recently been addressed to the isolation and characterization of the corresponding genes, since nitrate transport controls the amount of nitrogen assimilated by the cells(3) .
Nitrate transporter (NT) ()genes (nrtA, -B, -C, -D) have been cloned in both
unicellular and filamentous cyanobacteria and correspond to typical
bacterial binding protein-dependent transport
systems(8, 9, 10, 11) . The crnA gene from the filamentous fungus Aspergillus nidulans has
been proposed to encode for a nitrate
transporter(12, 13) . nar2, Nrt2;1,
and Nrt2;2 from the green alga Chlamydomonas reinhardtii have recently been identified as high affinity NT
genes(14, 15) . Nrt2;1 and Nrt2;2
were formerly named nar3 and nar4, respectively, and
their new names will be used hereafter to meet plant gene nomenclature
recommendations of the International Plant Molecular Biology
Society(16) . The Arabidopsis thaliana chl1 (Nrt1) gene is proposed to encode a low affinity NT (7, 17) and does not show significant homology with
any of the above-mentioned genes encoding high affinity
NT(10, 15) .
In C. reinhardtii, genes related to nitrate transport (nar2, Nrt2;1, and Nrt2;2) are located within a nitrate-regulated gene cluster containing the NR structural gene Nia1(14, 15, 18) . Mutants deleted in the nar2-NRT2;1-NRT2;2 genomic region are true NT mutants, which recover a high affinity NT activity upon transformation with plasmids carrying either nar2 plus Nrt2;1 or nar2 plus Nrt2;2 but not with any of these genes separately(15) . NRT2;1 and NRT2;2 proteins show a highly significant identity at the amino acid level with each other and with CRNA from A. nidulans(13) but not with any of the cyanobacterial nrt genes(10, 11) . CRNA, NRT2;1, and NRT2;2 are hydrophobic proteins that appear to have 12 hydrophobic membrane-spanning domains that would form the channel for nitrate(13, 15) . It has been suggested that NAR2 is either a structural or regulatory protein required for the function of NRT2;1 and NRT2;2, which could represent two alternative NT systems(15) .
In this work, NT mutants and transformed strains carrying different sets of nar genes have been molecularly and functionally characterized. Data strongly suggest that in C. reinhardtii there exist a nitrite-specific transporter independent of nar2 (system 3) and two additional NT systems: NAR2/NRT2;2 (system 2), which is specific for nitrate, and NAR2/NRT2;1 (system1), which efficiently transports both nitrate and nitrite.
Figure 1: Southern analysis of genomic DNA from the NT mutant S16 and transformant strains. A, restriction map of the genomic DNA region containing the nitrate assimilation gene cluster. Enzymes used are H, HindIII; K, KpnI; S, SstI; Sa, SalI; and Sm, SmaI. Approximate location and transcriptional orientation of nar, Nrt2, and Nia1 genes is shown by solid arrows(15) . C. reinhardtii DNA in plasmids pT1, pT2, and pB6a is indicated above the map. Probes B6a-6.2, a SalI-SstI genomic fragment, and 3`n4, a 360-base pair HindIII-EcoRV fragment at the 3`-non-translated region of Nrt2;2 cDNA, are indicated by solid lines. a, b, and c indicate expected fragments hybridizing these probes in Southern blots of genomic DNA digested with the indicated enzymes. B, genomic DNA (2-3 µg) isolated from the strains S16 (lane 1), 63-2 (lane 2), 63-4 (lane 3), 65-3 (lanes 4 and 6), and 65-4 (lanes 5 and 7) was digested with HindIII (lanes 1-3), SstI (lanes 4 and 5), and SmaI+KpnI (lanes 6 and 7) and analyzed by Southern hybridization with the indicated probes. C, genomic DNA (2-3 µg) isolated from strains S16 (lanes 1 and 8), 63-2 (lanes 4 and 6), 63-4 (lanes 5 and 7), 65-3 (lanes 2 and 9), and 65-4 (lanes 3 and 10) were digested with KpnI (lanes 1-5) and SmaI (lanes 6-10) and analyzed by Southern hybridization with the indicated probes.
Cells were grown at 25 °C under continuous light with 3-5%
CO-enriched air in ammonium minimal HS medium(20) .
Cells, induced in 4 mM KNO
media for 3
h(15) , were thoroughly washed and used for transport
experiments.
The copy number of introduced DNA in transformants was studied by digestions with enzymes that allow detection of bands with a restriction site in the genomic boundaries of the inserted plasmid DNA (Fig. 1C). Genomic DNA of strains 63-2 and 63-4 was digested with KpnI or SmaI and hybridized to the B6a-6.2 probe rendering unique bands of different sizes (lanes 4-7). This suggests that a single copy of pT2 integrated at different positions in the genome of each transformant. The pB6a flanking region was detected in 65-3 and 65-4 strains from KpnI digestions and hybridization with B6a-6.2. As shown in Fig. 1C, two hybridization bands were detected for transformant 65-3 and only one for 65-4 (lanes 2 and 3, respectively), which indicates again the presence of two copies of this B6a region in strain 65-3. The copy number of pT1 was studied by SmaI digestions of 65-3 and 65-4 genomic DNA and hybridization with the 3`n4 probe. Interestingly, two bands were detected for the 65-3 DNA and only one for 65-4 (Fig. 1C, lanes 9 and 10), which suggests that two copies of pT1 DNA are also present in the 65-3 genome.
On the other
hand, the stability through meiosis of the newly introduced genetic
markers in the transformant strains was studied by genetic crosses with
the wild-type 21gr strain (Table 1), considering that
the lack of either of nar2, Nrt2;1, Nrt2;2
or Nia1 leads to a Nia- phenotype (inability to grow on
nitrate minimal medium)(15) . Segregation ratio
Nia+:Nia- in the different crosses was very close to 2:1. A
segregation 9:7 should be found for unlinkage among all the studied
genes and 3:1 for linkage among all. The ratio found is close to 5:3,
which would be the expected one for the two nar2 and Nrt2 genes linked to each other and unlinked to Nia1
and the nitrate cluster region. In fact, nar2 and Nrt2;1 genes are physically linked in transformants with pT2
(strains 63-2 and 63-4), and it is known that Nia1
is unlinked to the gene cluster region in the parental strain
S16(15) . In addition, a loss of viability cannot be ruled out
in the segregations, since the S16 mutant was isolated from Nrt2; strain C2, which has a long deletion, and DNA
rearrangements occur in these mutant strains selected with
chlorate(14) . To study the existence of linkage in the newly
introduced DNA, four segregants from the cross 65-3
21gr and two from 65-4
21gr, having a
Nia- phenotype and an active NR enzyme, were selected. Southern
blot analysis of their genomic DNA showed that pT1 and pB6a plasmids
were absent (results not shown), which suggests that both genetic
markers are linked. It has been shown that plasmids integrated in
several copies in C. reinhardtii tend to be genetically linked (28) .
Figure 2:
Ammonium inhibition of nitrite uptake
activity by strains S16 and transformants with NT system 1, ) or NT
system 2,). Nitrate-induced cells were transferred to media for nitrite
uptake assay containing 25 µM KNO with (
)
or without (
) 1 mM NH
Cl, and nitrite in the
medium was measured at the indicated times.
Kinetic characterization of nitrite uptake and inhibition by nitrate has been studied in the C. reinhardtii wild type strain (29) . The results indicated that nitrate is a partially competitive inhibitor of nitrite uptake and seem to fit with the presence of different transporters for nitrate and nitrite in C. reinhardtii. Competition by nitrate and its analogue chlorate (30) on the nitrite transport by strains S16, 63-2, 63-4, 65-3, and 65-4 has been studied. As shown in Fig. 3, neither nitrate (50 µM) nor chlorate (1.5 mM) had an effect on nitrite uptake rates in strains S16 (carrying system 3) and 65-3 or 65-4 (carrying systems 2 and 3). However, both ions inhibited significantly nitrite uptake in strains 63-2 and 63-4 (carrying systems 1 and 3).
Figure 3:
Effect of nitrate and chlorate on the
nitrite uptake activity in S16, 63-2, 63-4, 65-3, and
65-4 strains. Nitrite uptake was determined as indicated in Fig. 2in media containing 10 µM KNO (control,
) and in the presence of 50 µM KNO
(
) or 1.5 mM KClO
(
).
The nitrite uptake inhibition by nitrate was also studied in the above strains by analyzing the effect of increasing nitrate concentrations on nitrite uptake, according to Córdoba et al.(29) . Data in Fig. 4show that nitrate concentrations up to 100 µM did not significantly affect nitrite uptake rates of system 3 determined at 10, 25, 50, and 75 µM nitrite in S16, 65-3, and 65-4 strains. In contrast, nitrate at µM concentrations strongly inhibited nitrite uptake rates of system 1 in strains 63-4 and 63-2. Analysis of these data are consistent with the existence of different transporters for nitrate and nitrite in C. reinhardtii(29) .
Figure 4:
Effect of different nitrate concentrations
on the nitrite uptake rate by S16, 63-2, 63-4, 65-3,
and 65-4 strains. Initial nitrite uptake rates were determined at
the KNO concentrations indicated in abscissae, in
the absence of nitrate (
) or in the presence of nitrate (10
µM,
; 25 µM,
; 50
µM,
; or 100 µM,
).
Kinetic parameters
for nitrate and nitrite transport in these strains have been determined (Table 2). In S16 strain, which only transports nitrite by system
3, K for NiT was 3.4 µM, the same
value as in strain 65-3, which has in addition the NT system 2.
In this strain, 65-3 nitrate was transported with a K
of 11 µM. In strain 63-4
carrying system 1, K
values were the lowest of
studied strains, 1.8 µM for nitrite and 1.6 µM for nitrate. Highest values of V
were also
obtained in strain 63-4. Kinetic parameters obtained for nitrite (29) and nitrate transport (Table 2) in the wild type
strain were very similar to those obtained in strain 63-4. V
for NiT in strain 65-3 was higher than
in S16. This reflects a better expression of the nitrite consuming
system 3 in strain 65-3 during nitrate induction, since the lack
of NT activity in S16 prevents an efficient induction of the system 3.
In fact, by increasing nitrate concentrations in the medium to 50
mM and higher, an induction of system 3 comparable to that in
the strains also carrying system 2 was achieved (not shown).
Data in this work strongly support a model in which three
different nitrate/nitrite transport systems operate in C.
reinhardtii. A protein carrier specific for nitrite is involved in
nitrite transport in the NT mutant strain S16, with an apparent K of 3.4 µM. This NiT activity seems
not to be due to a passive influx of nitrite since no diffusion
component was found under our experimental conditions ( (29) and this work). Nitrite uptake in C. reinhardtii was also blocked by ammonium in contrast to the cyanobacterial
passive nitrite transport, which is insensitive to
ammonium(31) .
The short-term blocking by ammonium of the nitrate/nitrite uptake is a well studied effect in C. reinhardtii and other organisms (1, 31, 32, 33) . However, its precise mechanism is still unknown. The inhibition is typically reversible, so the ability of the cell to utilize nitrate/nitrite is restored after ammonium is consumed from the medium. Both ammonium itself and a (some) product(s) of its metabolism seem to act together to block nitrite uptake in C. reinhardtii(33) .
The
protein carriers for NT identified in C. reinhardtii are
encoded by the crnA-homologous genes Nrt2;1 and Nrt2;2, with the requirement of a functional nar2
gene(15) . System 1 (NAR2/NRT2;1) appears to
mediate a high affinity and a high capacity transport specific for both
nitrate and nitrite, whereas system 2 (NAR2/NRT2;2) mediates a still
high affinity and specific NT, which showed a lower affinity and
capacity for nitrate than system 1. In C. reinhardtii,
chlorate sensitivity depends on chlorate concentration(30) .
Our results support that sensitivity to chlorate 1.5 mM is
mostly mediated by system 1. In Synechococcus sp. PCC 7942, nrtA, nrtB, nrtC, and nrtD are
responsible for a multicomponent transporter system that mediates
transport of both nitrate and nitrite(34) . In higher plants,
nitrate transport seems to be a more complex process; a biphasic
nitrate uptake kinetics has been reported, so that constitutive and
inducible systems might account for nitrate
transport(5, 6, 7, 17, 35) .
In addition, the existence of specific nitrite transporters is
uncertain(2, 3) . The clustered Nrt2/nar genes are ammonium-repressible and nitrate-inducible(34) ,
their expression requires an active product of the regulatory gene nit2(14, 37) , and according to our data they
encode for high affinity transporters specific for nitrate and nitrite.
The role of nar2 gene product seems to be specific for the NT
systems 1 and 2, since the lack of a functional nar2 gene
appears to have no effect on the regulation of the Nia1 gene
other than the lack of nitrate induction because of a deficient NT
activity(15) . ()The gene(s) responsible for the
specific nitrite transport in C. reinhardtii, which is
functional in strain S16, has not yet been isolated.
Nrt2;1
transcripts accumulate under different nutritional and physiological
conditions in amounts much higher than those of Nrt2;2, and
their amounts respond differentially to the culture growth
phase(36) . In this context, it is worth pointing out that the V found for the three transport systems studied
reflect how efficiently the corresponding system has been induced. In
S16, unable to take up nitrate, nitrate induction of the
ammonium-repressible system 3 specific for nitrite is not as efficient
as in strains carrying also the nitrate-specific transporter system 2.
Similar V
values were determined for two strains
carrying a different copy number of the nar2 and Nrt2;2 genes (molecular analysis suggests the presence of two
copies of pT1 and pB6a in 65-3, but only one in 65-4). If
both copies are functional, this might reflect a posttranscriptional
control of the nitrate transport machinery in C. reinhardtii. The response of these transporters to nutritional signals for
adjusting their relative amounts in the cell and their specific role in
promoting an efficient nitrate and nitrite transport are still open
questions.