• In plants, the knowledge of the molecular identity and functions of anion channels are still very limited, and are almost restricted to the large ChLoride Channel (CLC) family. In Arabidopsis thaliana, some genetic evidence has suggested a role for certain AtCLC protein members in the control of plant nitrate levels. In this context, AtClCa has been demonstrated to be involved in nitrate transport into the vacuole, thereby participating in cell nitrate homeostasis.
• In this study, analyses of T-DNA insertion mutants within the AtClCa and AtClCe genes revealed common phenotypic traits: a lower endogenous nitrate content; a higher nitrite content; a reduced nitrate influx into the root; and a decreased expression of several genes encoding nitrate transporters.
• This set of nitrate-related phenotypes, displayed by clca and clce mutant plants, showed interconnecting roles of AtClCa and AtClCe in nitrate homeostasis involving two different endocellular membranes.
• In addition, it revealed cross-talk between two nitrate transporter families participating in nitrate assimilation pathways. The contribution to nitrate homeostasis at the cellular level of members of these different families is discussed.
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In plant cells, electrophysiological analyses have revealed the presence of anion channels in most investigated membranes (plasma membrane, tonoplast, organelle membranes), where they participate in cell signalling, osmoregulation, plant nutrition and metabolism, and contribute to metal tolerance (Barbier-Brygoo et al., 2000; De Angeli et al., 2007). To date, very few of the associated proteins have been identified in Arabidopsis thaliana at the molecular level. Recently, at the plasma membrane, the SLAC1 protein has been shown to be an essential component of the slow-type anion channel in guard cells (Negi et al., 2008; Vahisalu et al., 2008). At the tonoplast, two anion transport systems have been clearly identified: the malate channel ALMT9 (Kovermann et al., 2007) and AtClCa, one of the seven members of the ChLoride Channel (CLC) family (De Angeli et al., 2006). The AtClCa protein acts as a /1H+ exchanger, a feature which supports the physiological characterization of a T-DNA insertion mutant in the AtClCa gene (clca-1), which displays an under-accumulation of nitrate in root and shoot tissues (by approximately 50%) and hypersensitivity to chlorate (Geelen et al., 2000). Three other members of the CLC family are targeted to endomembranes: AtClCe and AtClCf to thylakoid membranes and cis-Golgi vesicles, respectively, (Marmagne et al., 2007) and AtClCd to trans-Golgi vesicles (Von der Fecht-Bartenbach et al., 2007). Although some evidence of biological roles in agreement with the subcellular localization has been reported, direct evidence for the transport activity of these proteins could not be found in their native membranes, which are not easily amenable to electrophysiological techniques. In Arabidopsis, quantitative trait locus (QTL) analyses have provided a further genetic basis to correlate water and anion contents with nitrogen availability, driven by the hypothesis that nitrate acts osmotically to increase water uptake (Loudet et al., 2003), but the genes contributing to these QTLs are unknown. In a similar QTL analysis of nitrate storage, Harada et al. (2004) identified AtClCe, AtClCc and AtClCf as candidate genes contributing to quantitative variations in nitrate accumulation. Indeed, they showed that a clcc mutant displayed altered endogenous nitrate levels, demonstrating a role for at least one of these candidate genes. Together, these data suggest that AtCLC proteins participate in the control of the intracellular nitrate content in a manner which remains to be understood.
To gain an insight into the biological function of CLCs in plants, we focused on two members of the family, AtClCa and AtClCe. Previous data have demonstrated the role of AtClCa in vacuolar nitrate storage (De Angeli et al., 2006) and have suggested a putative role for AtClCe in controlling the ionic strength in the lumen of grana stacks in chloroplasts (Marmagne et al., 2007). In this article, we report further phenotypic analyses of T-DNA insertion mutants within the AtClCa and AtClCe genes, which reveal new nitrate-related traits in addition to nitrate under-accumulation already shown for clca (Geelen et al., 2000). We provide evidence for the interconnected contributions of the AtClCa and AtClCe proteins to nitrate homeostasis involving two types of endomembrane. We also show cross-talk between different families of nitrate transporters participating in the regulation of nitrate transport and assimilation pathways.
Materials and Methods
Plant material and culture conditions
Experiments were performed on several T-DNA insertion clc mutant lines in A. thaliana (L.) Heynh., two clca alleles in the Wassilewskija genetic background (WS), clca1 and clca2 (Geelen et al., 2000; De Angeli et al., 2006), and two clce alleles, clce1 (WS accession) and clce-2 (Columbia accession) (Marmagne et al., 2007). Selected homozygous lines were backcrossed using wild-type pollen. New homozygous mutant lines were then produced for phenotypic characterization in comparison with wild-type plants from the corresponding genetic background. The double mutants clca-1clce-1 and clce-1clca-1 in the reciprocal cross were produced and homozygous lines were selected. Sterilized seeds were grown in vitro on standard ABIS culture medium, as described by Geelen et al. (2000). clce1 mutant plants were complemented with the wild-type ClCe gene by agro-transformation with the pFP101-35S:ClCe construct [pFP101 was generated by Bensmihen et al. (2004) and kindly provided by F. Parcy, ISV, CNRS, Gif sur Yvette, France]. Two of nine independent transformants were selected for their high level of ClCe transcripts and analysed further.
RNA extraction and real-time PCR analyses
Total RNA samples were extracted from the different organs of adult plants grown for 2–6 wk on soil in the glasshouse and from total young plantlets or shoots and roots harvested from 10-d-old in vitro plantlets using the NucleoSpin RNA Plant kit (Macherey-Nagel, Düren, Germany). Real-time PCR was performed using the Light Cycler (Roche) on cDNA samples obtained with the SuperScript™ First-Strand Synthesis System for RT-PCR kit (Invitrogen) from 5 µg of total RNA. Oligonucleotides were identified using the program Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers for AtCLC genes were as follows: for AtClCa, aqS (5′-TCACACATCGAGAGTTTAGATT-3′) and aqAS (5′-AATGTAGTAGCCGACGGCGAGAA-3′); for AtClCb, bqS (5′-TCCCATATCGAAAGCTTGGACT-3′) and bqAS (5′-AGGAAGTGACCAACGGCTAAA-3′); for AtClCc, cqS (5′-GACGATGGATCTGTCGGATT-3′) and cqAS (5′-AAGCGCCCATTTGAGAAAC-3′); for AtClCd, dqS (5′-CGGAGGTGTCAATAGTCTCG-3′) and dqAS (5′-AATTTCCATCCAGCGAA-3′); for AtClCe, eqS (5′-GACGCTTGGGACTGGAAAT-3′) and eqAS (5′-CAACTGCAAAGAAGCATCCA-3′); for AtClCf, fqS (5′-CGTTGATGATCGAAATGAC-3′) and fqAS (5′-CTCTGTAGACGAAGCCATGC-3′); and for AtClCg, gqS (5′-TGCCAACGTCTGTCCAAT-3′) and gqAS (5′-ACTTAACACCGGCGAGATTC-3′). The primer sequences for the NRT1.1 gene are given in Richard-Molard et al. (2008), and for NRT2.1, NRT2.4, NRT2.5 and NRT2.7 are reported in Orsel et al. (2002). These primers amplified sequences of c. 200 bp in areas specific for the gene of interest and for the control actin28 gene. Each cDNA sample was tested with the primers in a series of three dilutions (400, 40 and 4 ng µl−1). Samples were subjected to 40 amplification cycles (PCR conditions as recommended in the Cyber-Green kit, Roche). After completion of the real-time PCR cycles, the results were analysed using the Light Cycler Software (Roche), and then normalised with the constitutively expressed actin28 gene.
Analyses of anion content
Capillary anion analysis was performed according to the procedure described by Geelen et al. (2000), with slight modifications. Basically, analyses were performed on c. 100 mg of 14-d-old plantlets grown on standard medium, harvested, ground in water and immediately freeze-thawed; the freezing–defreezing step was repeated three times. The ion analyser Millennium 2010 software (Waters, 78280 Guyancourt, France) was used for the quantification of various inorganic and organic anions according to freshly made standard solutions.
We also used a colorimetric assay for the measurement of the endogenous contents of nitrate and nitrite according to the original procedure of Barthes et al. (1995), with slight modifications. As the procedure measures the concentration, it is possible to obtain nitrite and nitrate contents in the same water extracts (prepared as described above). In the latter case, is converted into in the presence of exogenous nitrate reductase.
Nitrate influx measurements
The influx of 15 was assayed as described by Delhon et al. (1995). One or two plants grown in hydroponic culture, as described in Orsel et al. (2004), were transferred first to 0.1 mm CaSO4 for 1 min, and then to complete nutrient solution containing 0.2 mm15 (atom%15N: 99%) for 5 min, and finally to 0.1 mm CaSO4 for 1 min. Roots were separated from shoots immediately after the final transfer to CaSO4, dried for 48 h at 70°C and analysed using the ANCA-MS system (PDZ Europa, Crewe, Cheshire, UK). The influx of 15 was calculated from the 15N content of the roots [2 mg dry weight (DW)].
The clca and clce single mutants and the clca-1clce-1 double mutant under-accumulate nitrate and over-accumulate nitrite
We first characterized the clce-1 mutant, focusing on -related phenotypes displayed by young in vitro 14-d-old plantlets grown on 6 mm as sole nitrogen source. Analysis by capillary microelectrophoresis of inorganic and organic anion contents in whole clce-1 plants revealed that the level of nitrate, the most abundant anion detected, was reduced by 56% in mutant plantlets compared with the wild-type, whereas no significant alterations in the concentrations of chloride, sulphate and phosphate were found (Fig. 1). The levels of the organic acids citrate and malate were low, but the malate content was increased slightly by 1.5-fold in clce-1 tissues. A decrease in nitrate content, together with an increase in malate, was found in clce-1 root and shoot tissues from in vitro 14-d-old plantlets when analysed separately, although roots contained less nitrate than shoots (data not shown). These phenotypic traits are similar to those displayed by clca-1 mutant plants (Geelen et al., 2000).
Further analysis aimed to measure the nitrate and nitrite contents on the same extracts of homozygous mutant plants grown in vitro. Therefore, the different clca and clce mutant alleles were analysed in the same experiment. The clca mutants showed a stronger phenotype (c. −60% of endogenous nitrate) compared with the clce mutants (Fig. 2a). The double mutants, whatever the cross (clca-1clce-1 or the reciprocal clce-1clca-1), displayed a phenotype close to that of clca single mutants (Fig. 2a). Figure 2b illustrates that all mutant genotypes are also characterized by a higher endogenous content, clce mutants displaying the highest accumulation (+74% at most). The double mutants were characterized by a phenotype close to that of clce single mutants (Fig. 2b).
Furthermore, complementation of clce-1 plants by wild-type AtClCe cDNA resulted in a total restoration of both nitrate (Fig. 3a) and nitrite (Fig. 3b) wild-type endogenous contents in two independent transformed plants, confirming the link between the T-DNA insertion in the AtClCe gene.
AtCLC genes are differentially expressed in wild-type and clca and clce mutant plants
In quantitative real-time PCR experiments on cDNAs synthesized from mRNAs extracted from the organs of adult plants, very low AtClCa and AtClCe transcript levels were detected in tissues from adult plants compared with the level of actin mRNA (2–5%). Both AtClCa and AtClCe transcripts were ubiquitously detected all over the plant, but, in most organs, AtClCa expression was about ten times higher than that of AtClCe (Fig. 4a). The AtClCa gene was more strongly expressed in cauline and rosette leaves and flowers, but poorly in siliques, whereas AtClCe mRNAs were mainly present in leaves and in siliques. Analyses of mRNAs from aerial parts and roots of in vitro-grown plantlets showed that AtClCa transcripts were detected in shoots and roots, whereas the expression of AtClCe was restricted to the shoot (Fig. 4a). These data are in agreement with transcriptomics data available in Genevestigator (http://www.genevestigator.ethz.ch/at/) tools.
Compared with wild-type plants, the expression levels of AtClCb, AtClCc, AtClCd and AtClCf genes were increased 5–10-fold in clca mutant plants, whereas the AtClCe transcript level was strongly decreased (Fig. 4b). In clce plants, the gene expression pattern of all AtClC members was not significantly altered, except for a threefold increase in AtClCa transcript level, as shown in Fig. 4b, and in concordance with analyses performed on clce alleles in the Columbia background (data not shown).
Nitrate influx and expression of nitrate transporter genes are decreased in both clca and clce mutant plants
The nitrate influx capacities mediated by high-affinity nitrate transporters were compared in wild-type and mutant plants. For this purpose, nitrate influx was measured in the presence of a low 15 supply (0.2 mm) on roots from adult plants grown in hydroponic conditions (6 mm ). Compared with wild-type roots, roots from mutant plants showed a reduction in net nitrate influx by c. 40% in both clca and clce single mutants and in double mutants (Fig. 5A).
In the same series of experiments, the transcript levels of genes from the nitrate transporter families AtNRT1 and AtNRT2 (Orsel et al., 2002; Okamoto et al., 2003; Chopin et al., 2007) were evaluated. The AtNRT2.1 transcript level was strongly reduced in clca and clce mutant roots when compared with wild-type plants (Fig. 5Ba). For AtNRT2.4, AtNRT2.5 and AtNRT2.7 (Fig. 5Bb–d), no significant changes in transcript levels were observed, except for a slight decrease in AtNRT2.4 in clca roots and AtNRT2.5 in clce roots. By contrast, the expression of the AtNRT1.1 gene was strongly reduced in roots of both clca and clce mutant plants, but similar to wild-type levels in shoots (Fig. 5Be).
Two of seven members of the AtCLC family have been shown to be involved in nitrate assimilatory pathways: AtClCa (Geelen et al., 2000; De Angeli et al., 2006) and AtClCc (Harada et al., 2004). Knock-out mutant alleles in AtClCa and AtClCe genes (clca-1 and clca-2, Geelen et al., 2000; De Angeli et al., 2006; clce-1 and clce-2, Marmagne et al., 2007) provide interesting tools to investigate the integrated function of CLC proteins in plants. Homozygous clce-1 mutant plantlets under-accumulate nitrate, which is at least partially compensated by an increase in malate content, as already shown for clca1 (Geelen et al., 2000) and clcc mutants (Harada et al., 2004). The modifications of endogenous nitrate contents in clca and clce mutants were not associated with a change in nitrate reductase activity (data not shown). In addition, clca-1 and clce-1 mutants displayed an increase in nitrite content in all single and double mutant genotypes. We checked that, in clce mutants, nitrite reductase activity was not modified (data not shown). Together, our results show that both AtClCa and AtClCe participate in the different pathways involved in the regulation of free cellular nitrate content in plant cells (Crawford, 1995).
The AtClCa gene was expressed in shoots and roots, indicating the crucial role of AtClCa for vacuole nitrate accumulation not only in leaves (De Angeli et al., 2006), but at the whole-plant level, as suggested by nitrogen-related phenotypes in clca mutant roots. At the cellular level, in agreement with the anion transport function of AtClCa, the nitrate accumulated in the cytosol, in the absence of AtClCa-mediated vacuolar storage in the clca mutant (De Angeli et al., 2006), would be converted into nitrite, leading to endogenous nitrite accumulation as a secondary effect of the mutation. By contrast, the primary function of AtClCe located in thylakoids (Marmagne et al., 2007) has not been established directly to date. Indeed, AtClCe does not complement the yeast gef1 mutant (Marmagne et al., 2007). In other respects, it has not been possible to measure the transport activity by expressing AtClCe in heterologous systems, such as Xenopus oocytes or Sf9 insect cells (data not shown). Although pioneer studies have shown the presence of anion channels mediating chloride fluxes on thylakoid membranes of the higher plant Peperomia metallica (Schönknecht et al., 1988), and of the alga Nitellopsis obtusa (Pottosin & Shönknecht, 1995, 1996), no direct evidence for the presence of anion currents in Arabidopsis thylakoids has been provided so far. In a previous study, we have shown that clce mutants display a photosynthesis-related phenotype, in agreement with the higher AtClCe expression in green tissues compared with roots and, at the cellular level, with the thylakoid localization of the protein (Marmagne et al., 2007). This altered photosynthetic activity may result from an impaired anionic permeability of the thylakoid membrane. It seems reasonable to postulate a different anion selectivity for AtClCe compared with AtClCa because: (1) AtClCe is active in thylakoids, the inner membrane of chloroplasts supporting very specific biological functions, but not anion accumulation; and (2) the AtClCe amino acid sequence contains the serine residue (S160), which is part of the selectivity filter of ClC proteins sustaining chloride fluxes, instead of proline in AtClCa (Dutzler et al., 2002; Zifarelli & Pusch, 2009). In the chloroplasts, Cl− and, most probably, are good candidates as counter-anions able to compensate for the excess positive charges in the thylakoid lumen. In this situation, alterations in ionic strength or osmotic properties of chloroplast compartments may, in turn, directly or indirectly increase the nitrite level, most probably in the cytosol, as exhibited by both clce and clca mutants. This might suggest a role for AtClCe in nitrite translocation from the stroma into the thylakoids, taking over from the nitrite transporter of the chloroplast envelope (Sugiura et al., 2007).
This first set of data, supporting the role of AtClCa and AtClCe in nitrate assimilation pathways, raises the question of their coordinated role and of their possible interactions with nitrate transporters from NRT families. In roots, the AtNRT1.1 and AtNRT2.1 genes, encoding dual-affinity and high-affinity plasma membrane nitrate transporters (Liu & Tsay, 2003; Miller et al., 2007), act as major contributors in nitrate uptake (Miller et al., 2007). A close relation between the regulatory events affecting AtNRT1.1 and AtNRT2.1 gene expression was reported in the chl1-5 mutant, defective in the AtNRT1.1 gene (Muños et al., 2004). Furthermore, a 24-h treatment was shown to inhibit AtNRT1.1 transcript accumulation (Loquéet al., 2003). Indeed, in all clca and clce mutants, the reduced transcription of the nitrate transporter genes AtNRT1.1 and AtNRT2.1, and to a lesser extent AtNRT2.4 and AtNRT2.5, might result from the increase in endogenous nitrite. Interestingly, clca and clce mutants displayed the highest reductions in the high-affinity components of nitrate influx involving AtNRT2.1 and, partly, AtNRT1.1 transporters. A decrease in these components results, at least partly, from the observed effects at the transcriptional level. However, in our standard culture conditions (6 mm ), we observed reduced nitrate accumulation, suggesting that the AtNRT1.1 transporter probably contributes to the phenotype as a low-affinity component as well. Moreover, additional post-translational regulatory processes affecting nitrate transport systems cannot be excluded (Wirth et al., 2007). The transcript levels of AtNRT2.7, another member of the nitrate transporter family mostly contributing to nitrate loading into the vacuole during seed maturation (Chopin et al., 2007), were not modified in the clc mutants, suggesting that AtNRT2.7 might not have redundant functions with AtClCa, although also located in the vacuolar membrane. This set of data shows for the first time the existence of cross-talk between several transport systems involved in nitrate-related fluxes and responsible for the control of cytosolic nitrate homeostasis.
In the context of global nitrogen metabolism, it was surprising to observe the same physiological changes concerning nitrate transport and accumulation in clca and clce mutants, despite the fact that AtClCa is a nitrate transporter at the tonoplast and AtClCe is postulated to be a nitrite channel/transporter on thylakoid membranes. The analysis of transcript levels of the different ClCs in clca and clce mutants provided some clues to resolve this paradox. Indeed, both single mutations induce alterations in the expression pattern of other members of the CLC family. In particular, in clce mutants, the expression of AtClCa was slightly increased; by contrast, in clca mutants, a strong down-regulation of AtClCe expression was observed, leading almost to a double mutant clcaclce, in agreement with the fact that no additive effects for nitrogen-related phenotypes could be observed in the double mutant clca-1clce-1. On the basis of our hypothesis, these two different situations resulted in a common event, cellular increase, suggesting that a disruption/decrease in ClCe transport activity, in clce mutants and somehow in clca mutants, would be a major limiting step in nitrate utilization and storage. In clca and clce mutants, the increase in the endogenous content would, in turn, lead to the observed down-regulation of NRT gene expression, resulting in the observed decrease in nitrate influx and reduction in nitrate content.
Our data highlight the crucial role of AtClCe in nitrate assimilation through cytosolic nitrite, and raise the question of transcriptional links between AtClCa and AtClCe, which require further investigations. Nevertheless, the role of AtClCa in nitrate storage in mesophyll cells (De Angeli et al., 2006) as a function of provided nitrate (Geelen et al., 2000) is not questionable, although in clca mutants, part of the phenotype is a result of the down-regulation of AtClCe. Our results provide evidence that AtClCa and AtClCe, located in two different subcellular compartments, have interconnecting but not redundant functions in nitrate assimilation pathways.
We are grateful to B. Bernasconi and M. Vinauger-Douard (ISV, CNRS, Gif sur Yvette, France) for preliminary assays and quantitative PCR measurements, and to J.-M. Frachisse and F. Lelièvre (ISV, CNRS, Gif sur Yvette, France) for the endogenous anion content measurements by capillary electrophoresis. S. Oliva received financial support from Marie Curie (QLK5-CT-2001-60058). We are grateful to Pascal Tillard (BMP, Montpellier, France) for 15N measurements. We thank S. Filleur for critical reading of the manuscript. This project was supported by the Centre National de la Recherche Scientifique and the Génoplante programmes (AF1999035 and AF2001065). D. Monachello was funded by European Research Training Network NICIP CT-2002-000245.