Differential Regulation of the High Affinity Nitrite Transport Systems III and IV in Chlamydomonas reinhardtii *

Two high affinity nitrite transporters have been identified in Chlamydomonas reinhardtii. They have been named system III and system IV and shown to be differentially regulated by nitrogen and carbon supply. System III was induced under high CO2 and required a micromolar nitrate signal for optimal expression, was inhibited by ammonium, and was not affected by either chloride or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid. System IV was induced optimally under limiting CO2 and did not require nitrate signal, was inhibited by chloride and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but was not affected by ammonium. Two transcripts that shared the expression pattern of systems III and IV activities were detected with an Nrt2;3 gene probe. In addition, a mutant defective in both the activity of system III and the expression of Nrt2;3 gene has been isolated. Genetic crosses and in vivo complementation studies indicate that this mutant is defective in a locus that is closely linked to the regulatory gene Nit2.

Two high affinity nitrite transporters have been identified in Chlamydomonas reinhardtii. They have been named system III and system IV and shown to be differentially regulated by nitrogen and carbon supply. System III was induced under high CO 2 and required a micromolar nitrate signal for optimal expression, was inhibited by ammonium, and was not affected by either chloride or the chloride channel inhibitor 5-nitro-2-(3phenylpropylamino)benzoic acid. System IV was induced optimally under limiting CO 2 and did not require nitrate signal, was inhibited by chloride and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but was not affected by ammonium. Two transcripts that shared the expression pattern of systems III and IV activities were detected with an Nrt2;3 gene probe. In addition, a mutant defective in both the activity of system III and the expression of Nrt2;3 gene has been isolated. Genetic crosses and in vivo complementation studies indicate that this mutant is defective in a locus that is closely linked to the regulatory gene Nit2.
The first and key step of the nitrate assimilation pathway is the entry of nitrate into the cells mediated by specific transporters (1,2). Then, the sequential reduction of nitrate to nitrite, and nitrite to ammonium takes place in steps catalyzed by the enzymes nitrate reductase (NR) 1 and nitrite reductase (NiR), respectively (3)(4)(5).
NR and NiR genes are single copy in many algae, fungi, and plants (2,6,7). However, physiological and molecular data support the existence of redundant nitrate transporters (1,2). On the basis of nitrate affinity and expression conditions, transporters have been classified into constitutive or inducible high affinity transporters (CHANT, IHANT), and constitutive or inducible low affinity nitrate transporters (CLANT, ILANT). Molecular cloning of nitrate transporters from fungi, algae, and plants allows to classify them into two families (NRT1 and NRT2), according to sequence similarities (1). NRT1 belongs to the peptide transporter superfamily, and members of this family transport either nitrate or histidine with comparable efficiency (8). The Nrt2 genes belong to the major facilitator superfamily (1,9).
The Chl1 gene from Arabidopsis is the first member identified in the NRT1 family, which was primarily proposed to encode for a LANT regulated at the transcriptional level by nitrate and acid pH (10). Inducible LANT Chl1 analogues have also been identified in tomato, LeNrt1;2, whose substrate specificity is unknown (11), and Brassica napus, BnNrt1;2 which can transport nitrate and basic amino acids (8). Other members of the NRT1 family such as the Arabidopsis AtNrt1 (NTL1) and the tomato LeNrt1;1 are expressed constitutively (12), so they could correspond to the CLANT.
Nitrate assimilation is a highly regulated pathway in which nutritional and environmental conditions are determinant for metabolic adaptation. In this context, transporters should play an important role in regulating the amount of nitrate going into the cell according to its capability to assimilate or accumulate it. To know in a single organism each of the transporters, its specificity, and its regulation is a challenge to understand the nitrate assimilation pathway.
In C. reinhardtii, two HANT have been identified. The bispecific HANT/HANiT, named system I and encoded by Nrt2;1 and Nar2, and the specific HANT system II, encoded by Nrt2;2 and Nar2. Both are two-component systems which require for functionality a NAR2 protein (19,21). A third gene Nrt2;3 has recently been cloned, but its function remains to be demonstrated. Since deletion mutants lacking system I and II are able to assimilate efficiently nitrite, it has been proposed that Nrt2;3 could be responsible for nitrite transport (15).
In this work, two HANiT systems have been identified and shown to be differentially expressed depending on the nitrogen and carbon availability. Mutants deficient in nitrite transport activity and expression of the Nrt2;3 gene have been obtained. The possible regulatory role for the locus defective in one of these mutants is discussed.

EXPERIMENTAL PROCEDURES
Strains and Growth Conditions-C. reinhardtii wild type 6145c (mt Ϫ ), Nit2 mutant 203 (mt ϩ ), and deletion mutant D2 (mt Ϫ ⌬ Nrt2;2, Nrt2;1, Nar2, Nia1, Nar1) have been described elsewhere (14,22). Cells were routinely grown at 25°C under continuous light and 5% CO 2 -enriched (v/v) air in minimal HS medium containing 7.5 mM ammonium chloride (22). Cells were collected at mid-exponential phase of growth by centrifugation (4000 ϫ g, 5 min), washed twice with 50 mM potassium phosphate, pH 7.0, and then transferred to the indicated induction medium. Cultures were bubbled either with 5% CO 2 -enriched (v/v) air or with air washed through a saturated KOH solution. After the indicated times, cells were collected by centrifugation and processed immediately for enzyme assays, RNA extraction, or analytical determinations.
Genetic Procedures-Genetic crosses were carried out by the random spore plating method (23). Segregants analyzed for their ability to grow on media containing ammonium, nitrate, or nitrite. For in vivo complementation analysis, the mating mixture of gametes was plated in selective media containing 4 mM nitrate (24,25).
Enzyme Assays and Analytical Methods-Ammonium-grown cells or cells induced in different media were transferred to media containing 100 M nitrite, at a cell concentration of 10 -30 g Chl/ml. Samples were taken at different times, and nitrite concentration in the media was measured.
Nitrite was determined according to Snell and Snell (27), and chlorophyll as detailed by Arnon (28).
DNA and RNA Isolation from C. reinhardtii and Hybridization Analysis-Isolation of genomic DNA and Southern transfer were performed as previously reported (29,30). Conditions for hybridization were carried out according to Schloss et al. (31), and washes were performed at 65°C, with 0.2ϫ SSC and 0.2% SDS solution.
Radioactive probes were labeled by the random primer method (32). Probes used were: probe 1, a 1.1-kb KpnI-SacI fragment corresponding to the 5Ј region of the Nrt2;3 gene; probe 2, a XbaI-BamHI fragment corresponding to the 3Ј region of the Nrt2;3 gene; and probe 3, a KpnI-EcoRI fragment that contains the 5Ј region of Nrt2;3 gene in addition to the Nar5 gene (Ref. 15 and Fig. 3A).

Occurrence of Two Nitrite Transport Activities in Strain D2
from C. reinhardtii-The C. reinhardtii strain D2 has a deletion at the Nia1 genome region including Nrt2;2, Nrt2;1, Nar2, Nia1, and Nar1 genes. This strain is not deficient at the NiR locus and assimilates nitrite efficiently (14). Thus, this strain is useful for the study of potential nitrite transporters without the background of system I (Nrt2;1, Nar2) and system II (Nrt2;2, Nar2), previously identified (14,19).
Interestingly, the strain D2 grown in ammonium and transferred to nitrite medium was not able to take up nitrite under a 5% (v/v) CO 2 atmosphere, unless nitrate as a signal was present in the medium (Fig. 1A). The nitrate signal was sensed above 10 M, so that at 50 M nitrate induction of the nitrite transport activity was optimal. Since strain D2 cannot reduce nitrate to nitrite nor transport it efficiently under these conditions (14), the observed effect was considered as a nitrate signaling of nitrite transport activity induction. As shown in Fig. 1B, in the absence of CO 2 , strain D2 induced a nitrite transport activity without the requirement of nitrate signaling. These results seem to suggest, among other hypotheses, that there exist two different nitrite transporters, which are expressed differentially depending on the nitrate and carbon conditions. In order to verify this hypothesis, a further characterization was performed. These transport activities will be referred hereafter as system III, for the nitrate-signaled activ-ity under high CO 2 , and system IV, for the nitrate-independent activity induced under CO 2 deficiency.
The system III operated preferentially at pH 6 versus pH 8; meanwhile, system IV worked slightly better at pH 8 versus pH 6 under CO 2 deficiency (data not shown). These transport activities were not the result of nitrite diffusion coupled to a sink effect of the NiR enzyme. First, the nitrate signal was always needed for optimal expression of system III, even at pH 6 (data not shown). Second, NiR activity determined in cells induced under different nitrogen and carbon conditions was not the counterpart of the corresponding nitrite transport activity (Fig.  2). For example, in a medium containing 0.1 mM nitrite and bubbled with CO 2 , NiR activity was almost twice the activity observed without CO 2 , although nitrite was not consumed in the former condition.
The differential characteristics for these nitrite transport activities are summarized in Table I. System III transport activity was blocked by 1 mM ammonium, added as a sulfate or chloride form. This regulation by ammonium is a general characteristic of the nitrate/nitrite transporters identified until now in C. reinhardtii. So, when cells are consuming either nitrate or nitrite, addition of ammonium immediately blocks their transport activities (19,26,34). However, 1 mM ammonium did not affect the system IV activity when added as sulfate and had a slight inhibitory effect when added as chloride. This inhibition was due to chloride, since at 10 mM amounts of either NaCl or KCl, inhibition of system IV activity was much stronger. In addition, the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (35) inhibited system IV transport activity. In contrast, system III was slightly affected by chloride or NPPB. Systems III and IV were also regulated by CO 2 in a different way. System III transport activity was partially inhibited when CO 2 was removed from the medium. In contrast, system IV transport activity was blocked when cells were bubbled with 5% CO 2 .
Kinetic parameters were determined for both systems from the progress curves as previously reported (19,33). System III had a K s for nitrite of 5 Ϯ 2 M and a V max of 19 Ϯ 4 mol nitrite/h mg Chl. System IV had a K s for nitrite of 33 Ϯ 6 M and V max of 10 Ϯ 3 mol nitrite/h mg Chl. The K s for system III fits with that previously reported in C. reinhardtii (19). Northern blot analysis was performed to study expression of the Nrt2;3 gene in strain D2. Total RNA was isolated from cells subject to different nitrogen and CO 2 conditions. A KpnI-SacI 1-kb fragment corresponding to the 5Јregion of Nrt2;3 gene was used as a probe (probe 1, Fig. 3A). Interestingly, this probe recognized two transcripts of 2.8 and 2.4 kb, respectively, which were differentially expressed in strain D2 (Fig. 3B). This result suggests that in addition to the Nrt2;3 transcripts (15), another gene sharing significant homology with the used probe was detected. The expression pattern of both transcripts was different. At high CO 2 both transcripts were repressed in ammonium and optimally expressed when 100 M nitrate was present in the medium. However, when CO 2 was limiting the transcript of 2.8 kb was expressed under either nitrogen source, i.e. ammonium, nitrate, nitrite, or without nitrogen, although optimally in nitrite (Fig. 3B). When a XbaI-BamHI 4-kb fragment corresponding to the 3Ј region of Nrt2;3 gene was used as a probe (probe 2, Fig. 3C), the 2.4-kb transcript was mostly observed and their expression pattern fits with that obtained with the probe 1. That the 2.4-kb transcript was optimally expressed at high CO 2 , required a nitrate signal, and was almost absent in nitrite medium without CO 2 suggests a relationship between this transcript and system III transport activity. That the 2.8-kb transcript (named Nrt2;4) was optimally expressed under limiting CO 2 in a nitrate signalingindependent manner suggests that it could correspond to that of system IV.
Southern blot analyses were performed. DNA from strain D2 was digested with SacI, SmaI, and BamHI, and probed with the 5Ј region of Nrt2;3 KpnI-SacI fragment (Fig. 4). The most intense bands corresponded to the expected sizes for the Nrt2;3 gene region (15). Additional faint bands were found in digestions with the enzymes used, under high stringency conditions. The existence of these additional hybridization bands is in agreement with the presence of an Nrt2;3 analogue gene in strain D2.
Under high CO 2 and 0.1 mM nitrite, both transcripts were expressed (Fig. 3B, lane 4), NiR activity was high (Fig. 2), but no nitrite transport activity was detected (Fig. 1A). This seems to indicate that nitrate and/or CO 2 also have a regulatory role on the transporters at the post-transcriptional level. To gain more information, cells induced in the above medium during 5 h were processed in two ways. (a) Cells were kept with high CO 2 , and 0.1 mM nitrate, or nitrate plus cycloheximide were added to the medium (Fig. 5A). Then, nitrite transport activity was observed only when cycloheximide was not present, and with a kinetics much faster than in an induction from ammonium-grown cells. (b) Cells were bubbled with air lacking CO 2 in the presence or absence of cycloheximide, without nitrate signaling (Fig. 5B). Then, after CO 2 removal, only cells without cycloheximide showed nitrite transport activity. Again, this transport activity was significantly faster than in an induction from ammonium-grown cells. These results indicate that protein synthesis is required for expression of system III and IV transport activity in response to signaling by nitrate and absence of CO 2 , respectively.
Isolation of Nitrite Transport Mutants from Strain D2-In C. reinhardtii, nitrate/nitrite transporters have been shown to be responsible for chlorate toxicity. Although NR has a role in mediating chlorate toxicity, the absence/presence of NR activity is not critical for chlorate resistance/toxicity (2,36). Strain D2 is a chlorate-resistant mutant that lacks both HANT systems (I and II) and NR (14). When a functional NR is present in a D2 background, the resulting strain is also chlorate resistant (data not shown).
Since strain D2 contains other nitrite transporters, i.e. systems III and IV shown above, it could be possible to isolate nitrite transport mutants from this strain by selecting for chlorate-resistant colonies. However, strain D2 was resistant to 2 mM chlorate in medium containing a neutral nitrogen source such as urea. Notwithstanding, when 1 mM nitrate was present in the medium, strain D2 became sensitive to chlorate and spontaneous resistant colonies appeared in this medium at a ratio of about 1 ϫ 10 Ϫ6 . Twenty-two chlorate-resistant mutants were analyzed, and all were defective in activities and/or kinetic parameters of nitrite transport. Only one was stable, and its phenotype was consistent with that of a nitrite transport mutant. This strain named DC2-III had the following characteristics. (i) It did not grow in nitrite medium despite having NiR activity (data not shown). (ii) It lacked nitrite transport activity at high CO 2 (system III) (Fig. 6A). (iii) Under limiting CO 2 conditions, DC2-III strain induced a nitrite transport activity (Fig. 6B) that was presumably due to system IV, since it was inhibited by chloride, strongly by CO 2 , but not significantly by ammonium (Fig. 6C).
Molecular and Genetic Characterization of the Nitrite Transport Mutant DC2-III-Mutant DC2-III seems to be affected in a gene(s) involved in expression of nitrite transport system III. Nit2 from C. reinhardtii is the only positive regulatory gene identified in photosynthetic eukaryotes (25,37). Nit2 mutants have a Nit Ϫ phenotype and do not express NR or NiR (37)(38)(39). The Nrt2;3 gene is clustered with another nitrate regulated gene Nar5 whose function and relationship with nitrate assimilation are to be solved out. Nar5 expression does not seem to be under the control of Nit2 (15).
Nar5 and Nrt2;3-related genes were analyzed for expression in the strain DC2-III. Total RNA was isolated from ammoniumgrown or nitrate-induced cells for 3 h, under high CO 2 conditions. A KpnI-EcoRI fragment, which contains fragments of Nrt2;3 and Nar5 genes ( Fig. 3A; Ref. 15), was used as a probe. Results shown in Fig. 7 indicate that: (i) in contrast to the parental strain D2, both the 2.8-kb and 2.4-kb transcripts were almost undetectable in the strain DC2-III; and (ii) Nar5 expression was significantly decreased in DC2-III strain, when compared with that in the parental strain D2.
Since phenotype of mutant DC2-III was similar to that of Nit2 mutants, genetic analysis by in vivo complementation and genetic crosses was performed between these two strains. Diploid strains 203 (Nit2 Ϫ ) X DC2-III were able to grow in nitrateor nitrite-containing media. In addition, the genetic cross between strain 203 (Nit2 Ϫ ) and mutant DC2-III was analyzed from the growth of segregants in ammonium, and nitrate-or nitrite-containing media. The frequency of Nit ϩ /Nit Ϫ and Niit ϩ /Niit Ϫ segregants was 1:100, and 4:100, respectively. These results indicate that DC2-III was not defective at the Nit2 gene but in a locus that is closely linked to Nit2.

DISCUSSION
In this work, the existence of two HANiT systems (III and IV), in addition to the previously reported bispecific HANT/ HANiT system I and specific HANT system II (19), is demonstrated in C. reinhardtii. Physiological and molecular characterization of strain D2, which is deleted in the Nrt2;1 and Nrt2;2 genes encoding transport systems I and II (14), together with the isolation and characterization of the nitrite transport mutant DC2-III, allow us to assign particular characteristics to the two nitrite transport systems.
Systems III and IV were regulated differentially by nitrogen and carbon signaling. Expression of system III transport activity depends on a nitrate signal. This transporter operates at high CO 2 , and its activity is blocked by ammonium. In contrast, expression of system IV transport activity occurs at limiting CO 2 independently of signaling by nitrate. System IV activity is not inhibited by ammonium, but it is blocked by CO 2 , and strongly inhibited by chloride, or the chloride channel inhibitor NPPB. Two transcripts of 2.4 and 2.8 kb were detected from strain D2 in Northern blots at high stringency, and by using Nrt2;3 probes. The 2.4-kb transcript corresponded to the Nrt2;3 gene, whose previously reported transcript size (15) has been reevaluated with probes corresponding to either 5Ј or 3Ј regions of the gene. The 2.8-kb transcript was mostly detected with a probe from the 5Ј region of Nrt2;3. Sequence analysis of 5Ј region shows that Nrt2;3 is a member of the Nrt2 gene family (15), so the 2.8-kb transcript could correspond to a fourth gene TABLE I Effect of CO 2 , ammonium, chloride, and the chloride channel inhibitor NPPB on the nitrite transport activity by systems III and IV The nitrite transport activity of systems III and IV were induced for 3 h in nitrate media and high CO 2 and in nitrite media without CO 2 , respectively. Then, the nitrite transport activity was determined as indicated under "Experimental Procedures" by transfer of cells to fresh media containing 100 M NaNO 2 and the indicated compounds in the presence (ϩ) or absence (Ϫ) of CO 2 . 100% activity corresponded to 16  Ϫ at high CO 2 for 5 h. A, cells were kept at high CO 2 and 100 M nitrate with (q) or without (E) 1 g/ml cycloheximide was added to the media. B, cells were bubbled with air lacking CO 2 with (q) or without (E) 1 g/ml cycloheximide. Nitrite in the media was determined at the indicated times. of this family in C. reinhardtii, named Nrt2;4. The existence of a Nrt2;4 gene was also supported by the Southern blot data.
Systems III and IV could be related with Nrt2;3 and Nrt2;4 genes, respectively, since their corresponding transport activities show a similar expression pattern to each of these transcripts. The expression of the 2.4-kb/Nrt2;3 transcript was clearly dependent on the micromolar nitrate signal, meanwhile the expression of the 2.8-kb/Nrt2;4 transcript occurs when CO 2 was limiting, even in the presence of ammonium. In addition, in nitrite-containing medium at limiting CO 2 , where system IV activity is operative, the only transcript hybridizing to the Nrt2;3 probe was Nrt2;4.
The C. reinhardtii systems III and IV are HANiT with K s in the micromolar range. The characterization of C. reinhardtii NiR mutants has shown that there exist a LANT activity with a K s for nitrate in the millimolar range which appears to correspond to system III. 2 In addition, system IV in these NiR mutants shows a HANT activity with a K s for nitrate in the micromolar range. 2 No HANiT has been reported in higher plants until now. However, in C. reinhardtii, it can be summarized that the nitrate and nitrite transport systems correspond to the bispecific HANT/HANiT systems I and IV; and the specific HANT system II, and HANiT system III (Ref. 19 and this work). Systems I and II are essential in C. reinhardtii to support cell growth in nitrate-containing media (19,21). C. reinhardtii strains lacking systems I and II but having system III and IV are unable to grow in nitrate media, even though nitrate at low concentrations could be transported by system IV or at higher concentrations by system III (Refs. 14 and 19 and this work). Thus, the precise function of these systems III and IV in nitrate assimilation, other than transporting nitrite, remains to be solved. However, a regulatory role in controlling intracellular nitrate concentrations, according to nutritional and environmental conditions, could be suggested.
The nitrite transporter mutant DC2-III seems to be deficient in a locus, closely linked to Nit2, and involved in the nitrate signaling required for expression of system III/Nrt2;3. The selection strategy used to obtain this type of mutant is in agreement with this assumption. Parental strain D2 is chlorate-resistant in a neutral medium such as urea, since systems I and II are lacking (14), and systems III and IV are not operative under the high CO 2 conditions used for cell growth and selection. System IV is blocked by CO 2 , and system III requires a nitrate signal for optimal expression. Thus, by providing a nitrate signal, the NR-deficient D2 strain became sensitive to chlorate, which indicates that chlorate entry by system III was responsible for cell toxicity. The phenotype of mutant strain DC2-III, i.e. the absence of system III transport activity, the undetectable Nrt2;3 gene expression, and the significant decrease in the nitrate-dependent Nar5 gene expression, suggests that this strain is defective in nitrate signaling. Several hypotheses could explain these results. Mutant DC2-III could be defective in a regulatory gene mediating nitrate signaling by two different routes: one dependent on Nit2, which would switch on expression of nitrate assimilation genes (2,37), and another independent of Nit2, which would switch on Nar5 gene expression (15). Alternatively, the mutation could have affected the Nrt2;3 gene itself and thus the activity of system III, which would be responsible for nitrate signaling. In this context, it has been observed that the presence or absence of specific transporters in C. reinhardtii give different nitrate/ nitrite signaling effects. 3 Interestingly, genetic data showed that mutant DC2-III is defective in a locus closely linked to Nit2. Clustering of nitrate assimilation genes is appearing as a common feature in C. reinhardtii and might represent a strategy for efficiency in regulatory common mechanisms under very changeable environmental conditions. FIG. 6. Nitrite transport activities in the mutant strain DC2-III. A, induction of system III transport activity was performed as in Fig. 1A, either in the absence (E) or in the presence of 100 M nitrate (q). B, induction of system IV transport activity was performed as in Fig. 1B, either in the absence (E) or in the presence of 100 M nitrate (q). C, nitrite transport activity from cells previously induced for 5 h under limiting CO 2 was determined directly (E), or in the presence of 10 mM KCl (‚), 0.5 mM (NH 4 ) 2 SO 4 (q), and 5% CO 2 (OE).
FIG. 7. Expression of Nrt2;3 and Nar5 transcripts in the strain D2 and mutant DC2-III. A, total RNA isolated from strains D2 and DC2-III after 3 h in the presence of high CO 2 in media containing ammonium (A) or 4 mM nitrate (N) was hybridized with the probe 3 indicated in Fig. 3A. B, total RNA.