Download Nitrite transport to the chloroplast in Chlamydomonas reinhardtii

Journal of Experimental Botany, Vol. 53, No. 370,
Inorganic Nitrogen Assimilation Special Issue, pp. 845–853, April 2002
Nitrite transport to the chloroplast in Chlamydomonas
reinhardtii: molecular evidence for a regulated process
Aurora Galván, Jesús Rexach, Vicente Mariscal and Emilio Fernández1
Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Córdoba. Campus de Rabanales,
Edif. ‘Severo Ochoa’, 14071-Córdoba, Spain
Received 18 July 2001; Accepted 9 October 2001
Abstract
Nitrite transport to the chloroplast is not a well
documented process in spite of being a central step
in the nitrate assimilation pathway. The lack of
molecular evidence, as well as the easy diffusion of
nitrite through biological membranes, have made this
physiological process difficult to understand in plant
nutrition. The aim of this review is to illustrate that
nitrite transport to the chloroplast is a regulated step,
intimately related to the efficiency of nitrate utilization. In Chlamydomonas reinhardtii, the Nar1;1 gene
has been shown to have this role in nitrate assimilation. NAR1;1 corresponds to a plastidic membrane
transporter protein related to the bacterial formateu
nitrite transporters. At least four Nar1 genes
might exist in Chlamydomonas. The existence of
orthologous Nar1 genes in plants is discussed.
Key words: Chlamydomonas reinhardtii, nitrate assimilation,
nitrite transport.
Plastidic nitrite transport: a regulated process?
Compartmentation of different cell processes is known
to be a common strategy to enhance the efficiency of
the cell function. Compartmentation has an important
regulatory function in metabolism, mostly based on the
selective permeability of organelle membranes controlled
by specific transporters. This fact results in an increased
metabolic flexibility. Chloroplasts carry on key processes
in plants such as energy capture and storage, biosynthesis
of pigments, purines, pyrimidines, and fatty acids, and the
reduction of nitrite and sulphate (Buchanan et al., 2000).
1
Nitrite, when protonated, is reported to move easily
across biological membranes and this fact has hindered
the study of its putative specific transporters. In plant and
animal cells, anionic channels have been shown to be
permeable to both nitrate and nitrite (Barbier-Brygoo
et al., 2000; Frachisse et al., 2000; May et al., 2000;
Waldegger and Jentsch, 2000). In plants, anion channels
contribute to the regulation of a number of physiological
processes such as mineral nutrition, turgor- and osmoregulation, metal tolerance, and signal transduction
etc. (Schroeder, 1995; Cho and Spalding, 1996; BarbierBrygoo et al., 2000). Concerning nitrate assimilation,
chloride channels seem to play a role controlling the
intracellular nitrate homeostasis. In Arabidopsis thaliana
chloride channels have been shown to mediate a sustained
anion efflux in hypocotyl cells (Frachisse et al., 2000)
and, the disruption of a chloride channel (AtCLC-a) gene
results in a hypersensitivity to chlorate and a decreased
intracellular nitrate accumulation (Geelen et al., 2000).
Chloride channels have also been located in the envelope
membrane of chloroplasts and reported to be permeable
to nitrate and nitrite (Fuks and Hombé, 1999).
In addition to the chloride channels which are involved
in a number of different physiological processes, specific
transporters for nitrate assimilation are also involved. In
fact, several gene families corresponding to specific nitrate
assimilation transporters (from POT, MFS, and ABC
families) have been identified in fungi, yeast, algae,
plants, and bacteria, and reported to be involved in the
entry of nitrate into the cell (Crawford and Glass, 1998;
Moreno-Vivián et al., 1999; Forde, 2000; Galván and
Fernández, 2001).
In photosynthetic eukaryotic organisms, nitrate assimilation involves two membrane barriers, the plasma and
the chloroplast membranes. Thus, once nitrate is reduced
To whom correspondence should be addressed. Fax: q34 957 218591. E-mail: [email protected]
ß Society for Experimental Biology 2002
846
Galván et al.
to nitrite by the nitrate reductase enzyme in the cytosol,
nitrite has to cross the choroplast envelope membranes
for its subsequent reduction to ammonium and incorporation into amino acids (Hoff et al., 1994; Crawford,
1995). The idea of a controlled nitrite transport to the
chloroplast has been proposed by several authors
(Ritenour et al., 1967; Kessler et al., 1970; Brunswick
and Cresswell, 1988a, b; Krämer et al., 1988). However,
other work suggests that nitrite enters chloroplasts in its
protonated form by diffusion (Kaiser and Heber, 1983;
Shingles et al., 1996) and, more recently, that a chloride
channel located in the envelope membranes of chloroplasts is responsible for a passive transport of nitrate
and nitrite, but not of phosphate or sulphate (Fuks and
Homblé, 1999).
The requirement for a precise regulation of nitrite
transport into chloroplasts should be obvious. This step
seems to be key in plants and algae which use nitrate as
a nitrogen source, since it could limit the efficiency of
nitrate assimilation. In addition, nitrite is demonstrated
to be toxic and to induce oxidative stress. Nitrite is known
to produce DNA damage and oxidation of the cytochrome haem Fe2qto Fe3q (Derache, 1976; Zhao et al.,
2001); in erythrocytes nitrite induces methaemoglobin
formation and glutathione oxidation (Zavodnik et al.,
1999) and, in plants, nitrite inhibits photosynthesis and
CO2 fixation (Purczeld et al., 1978; Enser and Heber,
1980). The characterization of nitrite uptake by intact pea
chloroplasts provided evidence for a protein-regulated
process (Brunswick and Cresswell, 1988a, b) on the basis
of (1) the optimal alkaline pH, which would contrast with
a passive difussion of the protonated nitrite form, (2) the
lack of effect of anion channel inhibitors, (3) the saturation curve for nitrite, which raised a maximal activity
around 0.2 mM nitrite, and (4) the specific effect of
compounds which inhibit photosynthesis and either have
no effect on nitrite uptake or even stimulate it.
The Chlamydomonas reinhardtii Nar1;1
encodes a plastidic nitrite transporter
The study of functionality of the Chlamydomonas Nar1
gene supported the first molecular evidence for the
regulation of plastidic nitrite transport and the importance of this step in nitrate assimilation (Rexach et al.,
2000). Nar1, named Nar1;1 from now on (see below) is
clustered in chromosome IX together with genes encoding
nitrate reductase (Nia1), nitrite reductase (Nii1) and
the high affinity nitrate transporter (HANT) systems I
(Nrt2;1 and Nar2) and II (Nrt2;2 and Nar2) (Galván and
Fernández, 2001) (Fig. 1). Understanding the function of
Nar1;1 was a challenge because the redundancy of nitrite
transporters, also at the chloroplast membrane level,
results in no phenotype for Nar1;1-deficient mutants
Fig. 1. Nitrate assimilation gene cluster containing Nar1;1. The Nar1;1
region is shown in detail: the promoter ( ) containing hypothetical
nitrate regulatory elements (*), the 59 and 39 untranslated regions (h),
and the coding region with exons (E) and introns (I) (Rexach et al.,
2000). Positive and negative regulation is indicated as described in
the text.
under the standard laboratory growth conditions. The
function for Nar1;1, as a specific chloroplast nitrite transporter in nitrate assimilation, was assigned on the basis of
the following data.
(1) Like other genes in the nitrate cluster, Nar1;1
expression is under the control of the regulatory gene Nit2
(Quesada et al., 1993). Thus, Nar1;1 is expressed in
nitrate but not in ammonium media, which suggested a
role in nitrate assimilation (Fig. 1).
(2) The protein encoded by Nar1;1 corresponds to
an integral membrane protein predicted to have six
spanning-membrane domains, a plastidic localization
and significant identity to formate and nitrite transporters
from bacteria (Fig. 2) (Rexach et al., 2000).
(3) Nar1;1 allows nitrate utilization when this nutrient
is limiting for the cells. This limitation of nitrate takes
place in strains lacking the HANT systems I and II at
nitrate millimolar or, in strains having HANT, with
nitrate micromolar in the media (Rexach et al., 2000).
(4) The nitrite uptake activity by intact chloroplasts
isolated from Nar1;1q and Nar1;1 strains also supported that NAR1;1 is a plastidic nitrite transporter
with an apparent Ks for nitrite estimated at about 5 mM
(Rexach et al., 2000).
These data prompted the proposal that Nar1;1 defines
a structurally different and new type of transporter in
nitrate assimilation. This type of transporter is specific
for nitrite transport to the chloroplast and belongs to
the formate–nitrite transporter (FNT) family (Peakman
et al., 1990; Suppmann and Sawers, 1994; Saier, 1998).
The Nar1 gene family in Chlamydomonas
contains at least four members
The Chlamydomonas Nar1;1 gene sequence was used
to search for possible related sequences in the GenBank
databases (Stephen et al., 1997). Comparisons to Chlamydomonas ESTs from Kazusa DNA Research Institute
Nitrite transport to the chloroplast
Fig. 2. Predicted membrane topology of CrNAR1.1 plastidic membrane
protein. Membrane spanning domains were derived from the analysis
of multiple aligned CrNAR1;1 (Acc. No. AF149737), CrNAR1;2,
CrNAR1;3, CrNar1;4 (see legend of Fig. 3); EcFOCA (Acc. No.
P21501), and EcNIRC (Acc. No. P11097) sequences using the TMAP
program (Persson and Argos, 1994), taking into account hydrophobicity
profiles of these proteins (TMPRED program, http://www.ch.embnet.
org/software/tmbase/TMBASE_doc.html). This hypothetical model
shows residues conserved among CrNAR1 proteins in green, and in
blue conserved residues among CrNAR1 (sequences in Fig. 3) and
protein members of the FNT family (Acc. Nos: P21501, EcFOCA;
B82196, VcFOC; A39200, StNirC; P11097, EcNIRC; S39703, BsNiT;
A42712, MfFDH; F69974, BsFDH; CAA21934, CaHMP; and P38750,
ScYAH8). Acidic conserved amino acids are shown in yellow and basic
in red. Residues mostly conserved except for conservative substitutions
are indicated in lower case letters.
(http://www.kazusa.or.jp/en/plant/)
and
ChlamyDB
(http://www.biology.duke.edu/chlamy_ genome/)
have
allowed at least three new sequences of the NAR1 family
to be defined (Fig. 3). Thus, NAR1 has been renamed as
NAR1;1 and the other newly defined proteins NAR1;2 to
NAR1;5. However, NAR1;5 and NAR1;4 might correspond to the same protein, since no overlapping ESTs
were found (see the legend of Fig. 3).
Comparisons of the three NAR1 sequences at the
N-terminus with chloroplast envelope membrane proteins
(Brink et al., 1995; Weber et al., 1995; Kammerer et al.,
1998), and subsequent analysis by using Psort (http://
psort.nibb.ac.jpu; Nakai and Kanehisa, 1992), ChloroP1.1
(http://www.cbs.dtu.dk/services/ChloroP/; Emanuelsson
et al., 1999) and SignalP V1.1 (http://www.cbs.dtu.dk/
services/SignalP/; Nielsen et al., 1997) programs, allow a
plastidic location and a better definition of the putative
transit peptides to be assigned (Fig. 3). These NAR1
transit peptides have features of the chloroplast transit
peptides in Chlamydomonas, which differ from plants to
algae (von Heijne et al., 1989; Franzén et al., 1990) with
a typical high proportion of Ala, Ser and Arg residues.
However, transit peptides of envelope membrane
proteins differ structurally from thylakoid or stromal
proteins, and show a higher length of around 70–90
amino acids, with a small conservation among them and
with an N-terminal Ala residue (Brink et al., 1995; Weber
et al., 1995; Kammerer et al., 1998). Thus, NAR1;1 would
847
have a transit peptide of 82 residues, 10 amino acids
shorter than previously proposed (Rexach et al., 2000).
Putative NAR1;2 and NAR1;5 would have transit
peptides of 70 and 74 residues, respectively, and would
share with NAR1;1 a high proportion of Ala (25–45%)
and Pro (30–50%) in the last 20 amino acids (Fig. 3).
The Chlamydomonas ESTs present in the GenBank
databases do not have a single EST corresponding to
NAR1;1, but many from NAR1;2 and some of NAR1;3
to NAR1;5. This is clearly the result of the particular
conditions of mRNA expression from which the libraries
were constructed, which in turn might not be the optimal
for Nar1;1 expression. These ESTs come from libraries
constructed from cells subject to a wide range of culture
and stress conditions such as acetate containing media
and ammonium, or shifting to nitrate for 24 h, or
ammonium minimal medium at 5% CO2 and transfer
to 0.04% CO2, etc (see http://www.kazusa.or.jp/en/plant/
and http://www.biology.duke.edu/chlamy_genome/). The
putative mature NAR1 proteins show an identity from
31–49% among them, and around 30% with putative
formate and nitrite transporters from archea and
eubacteria. Members of the FNT family, which function
in the transport of structurally related anions (Peakman
et al., 1990; Suppmann and Sawers, 1994; Nolling and
Reeve 1997), do not appear to be present in cyanobacteria
and prochlorococcals. The FNT proteins contain around
270 residues, except for the yeast proteins (Accession
numbers P38750, and CAA21934), which show, in addition, a hydrophylic C-terminal extension of an unrelated
sequence and a variable length.
Phylogenetic analysis of proteins from the FNT family
has shown a clustering reflecting function and phylogeny
of the organism (Saier et al., 1998). As shown in the
dendogram of Fig. 4, NAR1;1 and NAR1;2 are closely
related, but distantly related to NAR1;3 and NAR1;4 and
to other bacterial nitrite transporters. Thus, the high
divergency of this family might make the identification
of orthologous genes in other eukaryotic species difficult.
Membrane topology of the FNT family members
is consistent with six putative transmembrane a-helical
spanning domains, as established for the FOCA protein
of E.coli (Suppmann and Sawers, 1994). Prediction of the
topology for the Chlamydomonas NAR1;1 protein is
consistent with the model shown in Fig. 2. Interestingly,
and according to this prediction, the protein would have
relatively short N- and C-terminus domains, with the first
five transmembrane segments bearing 26–29 residues
whereas the sixth domain would have 22 residues. As
indicated above, NAR1 sequences show a relatively low
conservation among them. Most of the conserved residues
lay within the second to the fifth transmembrane domains.
The signature proposed for the FNT corresponds to
the consensus patterns wLIVMAx-wLIVMYx-x-G-wGSTAxwDESx-L-wFIx-wTNx-wGSx in the second transmembrane
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Galván et al.
Fig. 3. Multiple alignment of the Chlamydomonas NAR1 predicted proteins. Predicted protein sequences correspond to CrNAR1;1 (Acc. No.
AF149737, Rexach et al., 2000), and the translated sequences from assembled Chlamydomonas ESTs as follows. CrNAR1;2: AV623045, BE727578,
AV628170, AV619728, BE725482, AV623679, AV619860, AV620323, AV622642, AV631000, AV622323, AV626567, AV622719, AV622426,
AV624687, AV631242, and BE211961; CrNAR1;3: AV644090, AV640494, and AV630068; CrNAR1;4: BF866634 and AV628342; and CrNAR1;5:
AV641981. Sequences were aligned using the DNASTAR program. Identical residues are indicated boxed in black. The putative start of mature
proteins is indicated with an arrow.
domain towards the third, and wGAx-x(2)-wCAx-NwLIVMFYWx(2)-V-C-wLVx-A within the fourth membrane segment (Suppmann and Sawers, 1994; Saier et al.,
1998). An additional highly conserved sequence pattern
for the FNT family within the fifth transmembrane
domain is proposed, F-wIVFAx-X-wLISx-G-wLFYTx-wEQxH-wSVCYx-wVIx-wAGx-wNDx-wMLQx.
Alignment of CrNAR1;1 with its closest relatives from
Chlamydomonas and the other members of the family
(Fig. 2) shows a high degree of co-linearity and conservation of these sequence patterns. The highest sequence
divergence appears in the hydrophilic segments of the proteins. Amino acid substitutions within transmembrane
domains are generally conservative, so that membrane
Nitrite transport to the chloroplast
q
Fig. 4. Dendogram of sequence relationships between CrNAR1 proteins
and selected members of the FNT family. The amino acid sequences
were aligned and the tree of sequence relationships was obtained by
using the Multialin program (http://w3.toulouse.inra.frumultalin.html;
Corpet, 1988). Accession numbers for the FNT sequences analysed
are: B82196 (VcFOC); C65025 (EcFOC2); A32305 (EcFOC); B65131
(EcNIT); P11097 (EcNIRC); A42712 (MfFDH); F69974 (BsFDH);
CAA21934 (CaHMP); S39703 (BsNiT); A39200 (StNirC); and P38750
(ScYH8).
topology is probably very similar in these FNT proteins
(Fig. 2).
The functionality of each of the NAR1 proteins will
need to be experimentally substantiated. As discussed
above, CrNAR1;1 is required for a plastidic HANiT
activity and for cell growth at limiting nitrate concentrations (Rexach et al., 2000). However, an additional
HANiT activity independent of NAR1;1 can be detected
in isolated chloroplasts from Chlamydomonas strains
deficient in NAR1;1, which could correspond to any of
the new NAR1 members identified (Rexach et al., 2000).
Also, members of this NAR1 family might correspond
to transporters for formate or another related anions. In
fact, the recent localization of formate dehydrogenase to
plastids in plants (Olson et al., 2000) would require the
activity of formate transporters in these organelles.
Nar1;1 and the efficiency of nitrate assimilation
An interesting phenotypic observation, when Chlamydomonas strains Nar1;1q and Nar1;1 were transferred
from high to low CO2, was the different ratio of TNRNA
(total nitrate reduced and non-assimilated). The term
TNRNA represents the sum of nitrite plus ammonium,
which are excreted to the media, from nitrate reduction at
849
limiting CO2 (Rexach et al., 2000). The Nar1;1 strains
were able to adapt to the situation of low CO2 and
excreted lower amounts of nitrite and ammonium from
nitrate than strains Nar1;1 . In other words, Nar1;1
appears to regulate nitrate reduction according to carbon
availability.
To understand the role played by Nar1;1 in nitrate
assimilation, comparative studies with Chlamydomonas
strains Nar1;1q and Nar1;1 have been performed under
three different conditions: (i) continuous light and high
CO2, (ii) continuous light and air, and (iii) lightudark
cycles and air. Nar1;1 does not confer any advantage for
a nitrate-dependent growth under continuous light and
high CO2, a laboratory condition which is not natural.
Under the second condition, continuous light and low
CO2, Nar1;1 gives a small advantage for nitratedependent growth. But, under the most physiological
condition, lightudark cycles and air, the presence of a
functional Nar1;1 gene provides a significant advantage
for the nitrate-dependent growth.
A working hypothesis is proposed to explain how
Nar1;1 facilitates nitrate assimilation in Chlamydomonas
(Fig. 5). This hypothesis is based on experimental data
from the evaluation of protein and starch content, the
expression of nitrateunitrite transporters and the activities
for nitrate and nitrite reductases in strains containing or
lacking Nar1;1 (V Mariscal et al., unpublished data). This
picture addresses the point that an apparent redundancy
of nitrateunitrite transporters is required for efficient
nitrate assimilation under different environmental conditions. At the plasma membrane, the four HANT systems
are represented: one specific for nitrate, system II (Nrt2;2,
Nar2); one specific for nitrite, system III (Nrt2;3); and two
bispecific for nitrate and nitrite, system I (Nrt2;1, Nar2)
and system IV (Nrt2.4?). At the chloroplast membrane,
NAR1;1 and the putative nitrite transporters identified
are represented.
When Chlamydomonas cells are grown in nitrate
media under lightudark cycles and low CO2, the presence
of Nar1;1 allows a regulated plastidic nitrite transport,
so that: (i) ammonium produced in the chloroplast will
be incorporated first by glutamine synthetase 2 (GS2),
(ii) since carbon skeletons are limiting, GS1, a cytosolic
form of GS (Chen and Silflow, 1996), would act by
recapturing the ammonium exported to the cytosol, and
(iii) low amounts of ammonium and nitrite, which have
not been incorporated, would finally be excreted to the
medium (Fig. 5A).
In Chlamydomonas cells lacking Nar1;1 and growing
under the same above conditions (Fig. 5B), the plastidic
nitrite transport will not be regulated accordingly with the
cell capability of incorporating ammonium into carbon
skeletons. After several lightudark cycles, overproduction of nitrite and mostly ammonium occurs so that:
(i) ammonium accumulated in the media blocks the
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Galván et al.
Fig. 5. Integration model for an efficient nitrate assimilation in Chlamydomonas mediated by NAR1;1. Metabolic integration involving transporters at
the plasma (PM) and chloroplast (CM) membranes in strains with (A) or without (B) a functional NAR1;1. Proteins involved are detailed and
explained in the text.
transport systems I, II and III; the system IV which is
insensitive to ammonium would be operative; (ii) at the
beginning of each new light period, elements of the
pathway which are negatively regulated by ammonium
and positively by light have to be expressed de novo
(Nrt2;1, Nrt2;2, Nar2; Nrt2;3 and Nia1; V Mariscal et al.,
unpublished data), but the presence of ammonium will
delay this expression. In addition, GS1 which is also
regulated by light and the nitrogen source (Chen and
Silflow, 1996) would be lower in Nar1;1 than Nar1;1q
strains, thus causing a less efficient incorporation of this
ammonium produced in the Nar1 strains.
The lack of a clear phenotype for Nar1;1 strains
under continuous light and high or low CO2 can be
Nitrite transport to the chloroplast
explained as follows: (i) at high CO2, there exists an
important sink of carbon skeletons where ammonium
produced from nitrate and nitrite reduction will be
incorporated, mainly by the plastidic glutamine synthetase GS2, and no ammonium would be excreted;
and (ii) at low CO2, the continuous light might ensure
a functional HANT system IV and GS1 to recapture the
nitrite and ammonium produced, respectively.
Thus, the lack of the plastidic nitrite transporter system
NAR1;1 would alter a regulated cross-talk between the
chloroplast and the cytosolic compartments, leading to
changes in the expression of HANT, nitrate and nitrite
reductase and probably GS1.
Are there plastidic NAR1 proteins in plants?
Comparisons of the CrNAR1;1 sequence with GenBank
databases resulted in the identification of an Arabidopsis
EST (Acc. No. N37972) which shows a 30% identity
at the protein level (Rexach et al., 2000). However, this
Arabidopsis EST does not match sequences from the complete Arabidopsis genome (http://www.arabidopsis.org/).
Additional EST fragments showing conservation with
the CrNAR1 proteins can also be found from Medicago
truncatula (Acc. No. BG451532), Triticum aestivum
(Acc. Nos BG312859, BG312420), and Sorghum bicolor
(Acc. No. AW287647). These sequences from Medicago
and Triticum correspond to E.coli FOCA genomic
sequence (Acc. No. P21501) raising the point on the
bacterial sequences contaminating eukaryotic libraries.
The sequence from Sorghum, which shows 41% identity
to NAR1;1 in 115 residues and smaller identities to
bacterial formate transporters, does not appear to
correspond to any sequence so far reported in the
GenBank.
The question as to whether or not NAR1 sequences are
conserved in plants needs further clarification. Two NAR1
orthologous sequences exist which could be candidates
for the corresponding protein in plants, one from
Arabidopsis, and another from Sorghum. However, this
very low number makes it necessary to substantiate their
existence. Other possibilities can be explored:
(i) Sequence conservation among NAR1 proteins from
plants and algae might not be high enough to allow an
easy identification of the orthologous proteins in plants.
As discussed above, except for CrNAR1;2, which shows a
49% identity to NAR1;1, the conservation among the
other CrNAR1 proteins is limited to about 30%.
(ii) In plants, the function of nitrite transport to
the chloroplast could be carried out by proteins unrelated
to the FNT family. In this respect, nitrate transport
is carried out by different protein families: ABC,
NRT2 and NRT1, whereas nitrite transport by ABC,
NRT2 and FNT proteins (Crawford and Glass, 1998;
851
Moreno-Vivián et al., 1999; Forde 2000; Galván and
Fernández, 2001; Moir and Wood, 2001).
In the GenBank database there are sequences from
cucumber (Acc. No. Z69370), Arabidopsis (within the
chromosome I, top arm complete sequence, Acc. No.
AE005172), and other plants which are referred to as
‘nitrite transporters’ to the chloroplast. In fact, analysis of
the sequence from cucumber by the ChloroP, Psort and
SignalP programs (see above) predicts a plastidic transit
peptide. Notwithstanding that there are no experimental
data supporting such a proposal, these sequences show
conservation with NRT1 from plants and members of
the proton oligopeptide family (Forde, 2000). Though the
existence of NRT1 proteins accounting for nitrite
transport to the choroplast is an atractive hypothesis,
there are no experimental data in support of this.
Concluding remarks
The unicellular eukaryotic green alga Chlamydomonas
appears to be a good model to unravel the complex
network of nitrate and nitrite transporters involved in
the compartmentalized nitrate assimilation pathway. The
functions of each transporter component are key for
understanding the correct balance of nutrients and the
signals required for a proper expression of genes and
proteins responsible for an efficient utilization of nitrate.
Although nitrite transport to the chloroplast has been a
step ignored until recently, molecular evidence indicates
that the Chlamydomonas Nar1;1 gene encodes a high
affinity nitrite transporter mediating an efficient nitrate
utilization. Nitrite transport to the chloroplast, like nitrate
transport at the plasma membrane level, is also the result
of the expression of different transporters, which might
correspond to the other NAR1 members. Since this transport step appears to be so critical in the fine regulation of
the whole pathway in Chlamydomonas, a similar situation
could be predicted in plants. However, the molecular
identity of these plastidic transporters in plants need to be
substantiated.
Acknowledgements
The authors thank MI Macias, I Molina and C Santos for their
technical and secretarial assistance. Funds were provided by the
EU Biotechnology program (BIO4C962231), the Dirección
General de Investigación Cientı́fica y Técnica, (PB98-1022CO2-02), and the Junta de Andalucı́a (PAI, CVI-128).
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