Journal of Experimental Botany, Vol. 53, No. 370, pp. 845-853,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
Nitrite transport to the chloroplast in Chlamydomonas reinhardtii: molecular evidence for a regulated process
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba. Campus de Rabanales, Edif. ‘Severo Ochoa’, 14071-Córdoba, Spain
Received 18 July 2001; Accepted 9 October 2001
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Abstract |
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 Top Abstract Plastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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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 formate/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.
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Plastidic nitrite transport: a regulated process? |
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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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
).
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; Barbier-Brygoo 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 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 Fe2+to Fe3+ (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.
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The Chlamydomonas reinhardtii Nar1;1 encodes a plastidic nitrite transporter |
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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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 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;1+ and Nar1;1- strains also supported that NAR1;1 is a plastidic nitrite transporter with an apparent Ks for nitrite estimated at about 5 µM (Rexach et al., 2000
).
- (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
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) containing hypothetical nitrate regulatory elements (*), the 5' and 3' untranslated regions (
), and the coding region with exons (E) and introns (I) (Rexach et al., 2000
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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).
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The Nar1 gene family in Chlamydomonas contains at least four members |
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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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 (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.jp/; 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 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 (seehttp://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.
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Membrane topology of the FNT family members is consistent with six putative transmembrane
-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 [LIVMA]-[LIVMY]-x-G-[GSTA]-[DES]-L-[FI]-[TN]-[GS] in the second transmembrane domain towards the third, and [GA]-x(2)-[CA]-N-[LIVMFYW](2)-V-C-[LV]-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-[IVFA]-X-[LIS]-G-[LFYT]-[EQ]-H-[SVCY]-[VI]-[AG]-[ND]-[MLQ].
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 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.
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Nar1;1 and the efficiency of nitrate assimilation |
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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An interesting phenotypic observation, when Chlamydomonas strains Nar1;1+ 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 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;1+ and Nar1;1- have been performed under three different conditions: (i) continuous light and high CO2, (ii) continuous light and air, and (iii) light/dark 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 nitrate-dependent growth. But, under the most physiological condition, light/dark 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 nitrate/nitrite 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 nitrate/nitrite 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.
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When Chlamydomonas cells are grown in nitrate media under light/dark 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 light/dark cycles, overproduction of nitrite and mostly ammonium occurs so that: (i) ammonium accumulated in the media blocks the 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;1+ 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 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.
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Are there plastidic NAR1 proteins in plants? |
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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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
; Moreno-Vivián et al., 1999; Forde 2000; Galván and Fernández, 2001; Moir and Wood, 2001).
- (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
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.
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Concluding remarks |
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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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.
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Acknowledgments |
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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 Científica y Técnica, (PB98-1022-CO2-02), and the Junta de Andalucía (PAI, CVI-128).
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Notes |
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1 To whom correspondence should be addressed. Fax: +34957218591. bb1feree{at}uco.es
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TopAbstractPlastidic nitrite transport: a...The Chlamydomonas reinhardtii...The Nar1 gene family...Nar1;1 and the efficiency...Are there plastidic NAR1...Concluding remarksReferences |
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