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Plant Physiol. Nov 2006; 142(3): 1075–1086.
PMCID: PMC1630733

Regulation of the High-Affinity NO3 Uptake System by NRT1.1-Mediated NO3 Demand Signaling in Arabidopsis[W]

Abstract

The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity equation M1 transport system (HATS) that plays a crucial role in equation M2 uptake by the plant. Although NRT2.1 was known to be induced by equation M3 and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when equation M4 concentration decreases to a low level (<0.5 mm) in media containing a high concentration of equation M5 or Gln (≥1 mm). The NRT3.1 gene, encoding another key component of the HATS, displays the same response pattern. This revealed that both NRT2.1 and NRT3.1 are coordinately down-regulated by high external equation M6 availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high equation M7 is specifically mediated by the NRT1.1 equation M8 transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up equation M9 in the presence of a reduced N source. Under low equation M10 provision, NRT1.1-mediated repression of NRT2.1/NRT3.1 is relieved, which allows reactivation of the HATS. Analysis of atnrt2.1 mutants showed that this constitutes a crucial adaptive response against equation M11 toxicity because equation M12 taken up by the HATS in this situation prevents the detrimental effects of pure equation M13 nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific equation M14 demand of the plant in relation to the various specific roles that equation M15 plays, in addition to being a N source. A new model is proposed for regulation of the HATS, involving both feedback repression by N metabolites and NRT1.1-mediated repression by high equation M16.

Higher plants acquire mineral nitrogen (N) from the soil mainly in the form of equation M17, through the activity of both high-affinity transport systems (HATS) and low-affinity transport systems (LATS), respectively (Crawford and Glass, 1998; von Wirén et al., 2000). Except in agricultural soils after fertilizer application, where equation M18 concentration can rise up several millimolar (Crawford and Glass, 1998), it is generally assumed that root equation M19 uptake is mostly determined by the activity of the HATS (Crawford and Glass, 1998; von Wirén et al., 2000; Malagoli et al., 2004). The current model of the equation M20 HATS is constituted by at least two genetically separate transport systems: (1) a constitutive HATS present even in the absence of equation M21 (Wang and Crawford, 1996; Crawford and Glass, 1998); and (2) an inducible HATS (iHATS), quantitatively more important than the constitutive HATS, and activated within hours after equation M22 provision to the plant (Behl et al., 1988; Mackown and McClure, 1988; Crawford and Glass, 1998; Daniel-Vedele et al., 1998; Forde, 2000).

In Arabidopsis (Arabidopsis thaliana), two gene families have been found to encode transporter proteins involved in root equation M23 uptake (Forde, 2000; Williams and Miller, 2001; Orsel et al., 2002; Okamoto et al., 2003): the NRT2 family (seven members) and the NRT1 family, belonging to the large PTR family of transporter genes (53 members). Convincing evidence has accumulated that the equation M24-inducible NRT2.1 gene encodes a major component of the iHATS (Filleur and Daniel-Vedele, 1999; Lejay et al., 1999; Zhuo et al., 1999). First, atnrt2.1 mutants display a strong reduction of root equation M25 influx in the low external equation M26 concentration range (i.e. below 1 mm; Filleur et al., 2001; Orsel et al., 2004). Second, equation M27 inducibility of high-affinity root equation M28 uptake is suppressed by NRT2.1 deletion (Cerezo et al., 2001). Recently, another key component of the equation M29 HATS has been identified in the form of the product of the NAR2-like Arabidopsis gene NRT3.1 (Okamoto et al., 2006). It is suspected that interaction between NRT2.1 and NRT3.1 is required for functionality of the iHATS because NRT2 proteins need to be coexpressed with NAR2-like proteins to generate equation M30 uptake activity in Xenopus oocytes (Quesada and Fernandez, 1994; Zhou et al., 2000; Tong et al., 2005).

Besides induction by equation M31, NRT2.1 expression and equation M32 HATS activity are also feedback repressed by reduced N metabolites, such as equation M33 and amino acids (Lejay et al., 1999; Zhuo et al., 1999; Nazoa et al., 2003). This regulation involves systemic signaling (Gansel et al., 2001) and ensures the specific control of root equation M34 uptake by the N status of the whole plant (Imsande and Touraine, 1994; Forde, 2002). Indeed, N sufficiency triggers the repression exerted by N metabolites and down-regulates the HATS, whereas N deprivation has the opposite effect (Crawford and Glass, 1998; Lejay et al., 1999; Zhuo et al., 1999; Gansel et al., 2001). Again, NRT2.1 plays a key role in these processes because both repression of equation M35 HATS by N metabolites and its stimulation by N starvation are suppressed in the atnrt2.1-1 mutant (Cerezo et al., 2001). Another important aspect of NRT2.1 function relates to the recent reports showing that it is not only involved in high-affinity equation M36 uptake, but also plays a equation M37-sensing role in the regulation of lateral root initiation (Little et al., 2005; Remans et al., 2006). This latter role is still unclear because, depending on the conditions, NRT2.1 either represses (Little et al., 2005) or, on the contrary, stimulates (Remans et al., 2006) lateral root development.

Recently, investigation of several chl1 mutants of Arabidopsis unexpectedly revealed that disruption of another equation M38 transporter gene, NRT1.1 (formerly CHL1), results in a major alteration of the regulation of NRT2.1 expression by the N status of the plant (Muños et al., 2004). First, feedback repression of NRT2.1 by either equation M39 or Gln supply is suppressed or strongly attenuated in chl1 mutants compared with wild type, resulting in its overexpression in normally suppressive conditions (e.g. in NH4NO3-fed chl1 plants). Second, expression of NRT2.1 is no more stimulated by N starvation in chl1 mutants. These data suggest that mutation of NRT1.1 blocks both NRT2.1 expression and equation M40 HATS activity in some kind of derepressed state, making chl1 mutants the only known genotypes affected in the regulation of root equation M41 uptake in higher plants.

NRT1.1 is an unusual dual-affinity equation M42 transporter (Tsay et al., 1993; Wang et al., 1998), shifting from low to high affinity in response to phosphorylation of the Thr-101 residue (Liu and Tsay, 2003). Although NRT1.1 also contributes to root equation M43 uptake (Tsay et al., 1993; Huang et al., 1996; Touraine and Glass, 1997), its precise role in the regulation of NRT2.1 expression remains unclear. A first hypothesis was that NRT1.1 may be directly involved in the signaling pathway responsible for feedback repression of NRT2.1 by N metabolites. However, this interpretation was questioned by the finding that repression of NRT2.1 by equation M44 could also be alleviated in wild-type plants when the external equation M45 concentration was decreased down to 0.1 mm in the presence of 1 mm equation M46 (Muños et al., 2004). This showed that mutation of NRT1.1 is not required to suppress the repressive effect of N metabolites and led to a second alternative hypothesis that NRT2.1 up-regulation in NH4NO3-fed chl1 plants could actually be due to the lowered equation M47 uptake resulting from NRT1.1 mutation. Because NH4NO3-fed chl1 plants were not N deficient, this in turn suggested that NRT2.1 expression is not only repressed by reduced N metabolites, but also by equation M48 itself (Muños et al., 2004).

This work had three aims: (1) to investigate the occurrence of such repression of NRT2.1 by equation M49 in wild-type plants; (2) to determine whether NRT1.1 plays a direct (i.e. specific) or indirect (through modulation of equation M50 uptake rate) role in triggering this repression; and (3) to determine the physiological role of this regulation. Concerning this last point, we hypothesized that repression of NRT2.1 by equation M51 may correspond to a mechanism allowing the HATS to be stimulated by a specific equation M52 demand of the plant independently of its overall N status (Muños et al., 2004). In particular, we anticipated that this may have a crucial function to avoid that the bulk of N acquisition from mixed NH4NO3 medium is made through equation M53 uptake, thus protecting the plant against the toxicity generally associated with equation M54 nutrition (Givan, 1979; Hageman, 1984; Salsac et al., 1987; Kronzucker et al., 2001).

RESULTS

Low NO3 Availability in the Presence of NH4+ Up-Regulates Both Root NRT2.1 Expression and NO3 HATS Activity

As commonly observed in many experimental conditions, the supply of equation M55 together with equation M56 at equimolar concentration (1 mm for both ions) is associated with very faint expression of NRT2.1 in the roots (Fig. 1A, lane T0). However, transfer of plants to fresh nutrient medium with lower equation M57 concentration (0.1 mm), but unmodified equation M58 availability (1 mm), rapidly resulted in a marked increase in NRT2.1 mRNA accumulation, which was detectable after 6 h and leveled off between 3 and 4 d (Fig. 1A).

Figure 1.
Changes in NRT2.1 mRNA accumulation in response to various equation M292/equation M293 mixtures in the external medium. Plants were grown hydroponically for 6 weeks on complete nutrient solution containing 1 mm NH4NO3 before transfer to the various media indicated in the figure. ...

To identify more precisely which parameter modified by the above treatment (equation M59 concentration, total equation M60 plus equation M61 concentration, or equation M62 to equation M63 ratio) was responsible for the up-regulation of NRT2.1 expression, we explored the effects of various combinations of equation M64 and equation M65 concentrations. Figure 1B displays typical results obtained from this series of experiments. They show that the increase of NRT2.1 transcript accumulation is not due to the lowering of the total external N (equation M66 + equation M67) concentration because it was also observed after transfer to solutions containing 0.1 mm equation M68 supplemented with 2 mm, or even 5 mm equation M69. On the other hand, a high equation M70 to equation M71 ratio in the external medium is also not responsible per se for up-regulation of NRT2.1 expression because, when compared to 1:0.1 mm equation M72:equation M73, the 5:0.5 mm equation M74:equation M75 mixture (with the same equation M76:equation M77 ratio of 10) resulted in much less pronounced NRT2.1 up-regulation (Fig. 1B). Furthermore, NRT2.1 is also overexpressed by low equation M78 availability in mixed N medium when Gln (another potent repressor of NRT2.1 expression) is provided as the reduced N source (Supplemental Fig. S1). This shows that NRT2.1 up-regulation is not specifically due to excess equation M79 supply. Altogether, the data obtained suggest that NRT2.1 expression is responsive to the absolute equation M80 concentration present in the mixed N solution. Indeed, whatever the nature and concentration of the reduced N source (equation M81 or Gln, 1–5 mm), the NRT2.1 transcript level was always high when the external equation M82 concentration remained low at 0.1 mm, but strongly decreased when the equation M83 concentration was raised at 0.5 mm.

One interesting finding related to the increased expression of NRT2.1 under low equation M84/high equation M85 availability is that the effect is local and not systemic. Indeed, split-root experiments showed that when only one side of the root system is provided with 0.1 mm equation M86 + 1 mm equation M87, NRT2.1 is up-regulated in this side only and not in the other one fed with 1 mm NH4NO3 (Fig. 2, compare lanes b and c). Furthermore, the NRT2.1 transcript level in the side of the split-root system supplied with 0.1 mm equation M88 + 1 mm equation M89 (Fig. 2, lanes c and e) was the same, whatever the equation M90 availability on the other side (1 or 0 mm in Fig. 2, lanes b and f, respectively), and equaled that in plants homogeneously supplied with 0.1 mm equation M91 + 1 mm equation M92 on the whole root system (Fig. 2, lane d).

Figure 2.
Effect of local changes in the equation M298/equation M299 external balance on NRT2.1 mRNA accumulation in split-root plants. A, Representation of the experimental protocol. Plants were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mm NH4NO3. After ...

In wild-type plants, up-regulation of NRT2.1 expression by low equation M93 availability in the presence of equation M94 resulted in similar stimulation of equation M95 HATS activity (as measured by root 15equation M96 influx at 0.2 mm), which was not observed in the atnrt2.1-1 mutant (Fig. 3A). Interestingly, the equation M97 uptake system did not display this stimulation because root 15equation M98 influx measured either at 1 mm (Fig. 3B) or 0.2 mm (data not shown) was only marginally increased upon lowering of the external equation M99 concentration. Total N content of roots and shoots was unaffected between the treatments (data not shown), most probably as a result of both increased equation M100 HATS activity and sustained equation M101 uptake.

Figure 3.
Effect of various equation M302/equation M303 mixtures on NRT2.1 mRNA accumulation and root N uptake in Ws wild-type and atnrt2.1-1 mutant plants. The plants were grown hydroponically for 6 weeks on complete nutrient solution containing 1 mm NH4NO3 before transfer for 4 d to various ...

Collectively, the above results suggest that low equation M102 availability alleviates repression of both NRT2.1 expression and equation M103 HATS activity triggered by provision of a reduced N source to the plant.

Regulation of NRT2.1 Expression by External NO3 Availability in the Presence of NH4+ Is Specifically Mediated by NRT1.1

In previous experiments with chl1-10 plants, it was observed that increasing equation M104 concentration from 0.1 to 10 mm in the presence of 1 mm equation M105 failed to repress NRT2.1 expression (Muños et al., 2004), unlike what is found in wild type (Fig. 3A). To determine whether this is due to a direct or an indirect (through lowered total equation M106 uptake) role of NRT1.1 in regulating NRT2.1 expression, we checked whether the mutation of NRT1.2, encoding another important component of the equation M107 low-affinity transport system (Huang et al., 1999), also resulted in NRT2.1 up-regulation. Therefore, we investigated the quantitative relationship between equation M108 uptake from NH4NO3 medium and NRT2.1 expression in the roots of three genotypes: the Wassilewskija (Ws) wild type, the chl1-10 mutant, and the atnrt1.2-1 mutant, carrying a T-DNA insertion in the NRT1.2 gene.

Increasing the external equation M109 concentration from 0.1 to 5 or 10 mm in the presence of 1 mm equation M110 resulted in a strong and almost similar down-regulation of NRT2.1 expression in both Ws and atnr1.2-1 plants, whereas this down-regulation was absent in chl1-10 plants (Fig. 4A). Surprisingly, root 15equation M111 influx (measured at the same concentration as that used for treatment of the plants) was little affected by external equation M112 concentration in Ws plants (Fig. 4B), where a 50-fold decrease of this concentration (from 5–0.1 mm) only resulted in a 30% slowing down of root 15equation M113 influx. This indicates strong homeostasis of root equation M114 uptake from NH4NO3 medium, which probably explains why root equation M115 influx was only marginally stimulated by the decrease in external equation M116 concentration (Fig. 3B). Root 15equation M117 influx was lower in the atnrt1.2-1 mutant than in the wild type, but not specifically in the high external equation M118 concentration range. Unexpectedly, root 15equation M119 influx was not reduced in chl1-10 plants compared with Ws and was even higher in the middle range of external equation M120 concentration (1–5 mm). These data unambiguously demonstrate that unrepressed NRT2.1 transcript accumulation in chl1-10 plants at high external equation M121 availability cannot be explained by reduced root equation M122 uptake rate as compared to either Ws or atnrt1.2-1 plants (Fig. 4C). Thus, whatever the signal responsible for down-regulation of NRT2.1 expression by high external equation M123 availability, it requires NRT1.1 to trigger the response.

Figure 4.
Relationship between NRT2.1 mRNA accumulation and root equation M312 influx as a function of equation M313 concentration in the presence of equation M314 in the external medium. Plants of Ws wild-type, atnrt1.2-1 mutant, and chl1-10 mutant were grown for 6 weeks on complete nutrient solution ...

Additional evidence for NRT1.1-dependent control of the equation M124 HATS by external equation M125 availability was provided by expression analysis of NRT3.1, which shows the same response pattern as NRT2.1, suggesting coregulation of the two genes (Fig. 5). However, the quantitative changes in NRT3.1 transcript abundance were generally less pronounced than those of NRT2.1.

Figure 5.
NRT2.1 and NRT3.1 coregulation. A, Response of NRT3.1 mRNA accumulation in Ws, atnrt1.2-1, and chl1-10 plants to the variation of equation M321 concentration in the presence of 1 mm equation M322 in the external medium. The experiment is the same as in Figure 5. B, Correlation ...

Taken together, the above data strongly support the hypothesis of coordinated regulation of NRT2.1 and NRT3.1, mediated by NRT1.1, which down-regulates the equation M126 HATS when external equation M127 availability in mixed N medium is above 0.2 to 0.5 mm. Below this threshold, this repression is alleviated, which enables the plant to reactivate the equation M128 HATS, despite the strongly repressive conditions related to the ample supply of reduced N source.

Up-Regulation of NRT2.1 by Low NO3 Availability in the Presence of NH4+ Prevents Growth Inhibition Associated with NH4+ Toxicity

To determine the physiological significance of NRT1.1-dependent control of the equation M129 HATS by external equation M130 availability, we investigated the hypothesis that up-regulation of NRT2.1 by low equation M131 concentration in mixed N medium plays a role in preventing equation M132 toxicity under situations of excess equation M133 over equation M134 supply. Therefore, we analyzed growth and equation M135 uptake of wild-type plants and of two independent NRT2.1 knockout mutants (atnrt2.1-1 and atnrt2.1-2) under pure equation M136 or mixed NH4NO3 nutrition. Seedlings were first grown for 5 weeks on 1 mm NH4NO3 before submitting them for 10 to 14 d to the various N nutrition regimes. During these experiments, wild-type and mutant plants were placed in the same container to make sure that both genotypes experienced the same changes in external pH.

As expected, the supply of 1 mm equation M137 as the sole N source led to the appearance of toxicity symptoms in the shoots of both wild-type and atnrt2.1-1 genotypes (Fig. 6). These symptoms became pronounced between 6 and 10 d after transfer to the equation M138 nutrient solution. In particular, equation M139-fed plants started to bolt very early and their leaves wilted and yellowed. Interestingly, addition of 0.1 mm equation M140 in the 1 mm equation M141 nutrient solution fully prevented the appearance of toxicity symptoms in wild-type plants, but not in the mutant, which seemed to remain as sensitive as when equation M142 is the sole N source supplied (Fig. 6).

Figure 6.
Effect of NRT2.1 mutation on the protective effect of equation M323 against equation M324 toxicity. Images show the appearance of toxicity symptoms in shoots of Ws and atnrt2.1-1 mutant plants as a function of the N source supplied in the external medium (either 1 mm equation M325 alone or ...

These observations were confirmed by shoot growth analysis (Fig. 7A). With 1 mm equation M143 as the sole N source, shoots of atnrt2.1-1 plants grew at the same relative rate as those of wild-type plants, despite slightly lower biomass at the beginning of the experiment. Relative growth rate (RGR) values (determined from the slopes of the linear relationships between ln [fresh weight] and time) were 0.126 g g−1 d−1 (r2 = 0.986) and 0.125 g g−1 d−1 (r2 = 0.975) for wild-type and atnrt2.1-1 shoots, respectively. Supply of 0.1 mm equation M144 together with 1 mm equation M145 markedly stimulated shoot growth in wild-type plants (RGR = 0.165 g g−1 d−1; r2 = 0.992), but not in atnrt2.1-1 plants (RGR = 0.137 g g−1 d−1; r2 = 0.988). In agreement with the fact that NRT2.1 is up-regulated by low equation M146 concentration in mixed N medium, and that it encodes a major component of the equation M147 HATS, the cumulative equation M148 uptake in wild-type plants transferred on 0.1 mm equation M149 + 1 mm equation M150 was much higher than that in atnrt2.1-1 plants (Fig. 7B). All the above observations were confirmed with the atnrt2.1-2 mutant allele (Supplemental Fig. S2). On 1 mm NH4NO3, however, a situation where NRT2.1 has a low contribution to total equation M151 uptake (Cerezo et al., 2001), no significant difference was recorded for both shoot biomass and cumulative equation M152 uptake between wild-type and atnrt2.1-1 plants (Supplemental Fig. S3). Thus, the decrease in shoot growth observed in atnrt2 mutants as compared to wild types correlated with the reduction of equation M153 uptake resulting from the NRT2.1 mutation. On the other hand, root biomass was not affected by NRT2.1 mutation in all media investigated (Supplemental Figs. S2 and S3).

Figure 7.
Shoot growth and root equation M327 uptake of Ws and atnrt2.1-1 mutant plants supplied with 1 mm equation M328 with or without 0.1 mm equation M329. Plants were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mm NH4NO3 before transfer for 12 d to media containing ...

Taken together, the above data demonstrate that, even at a low concentration of 0.1 mm, the presence of equation M154 in mixed N solution is able to alleviate the detrimental effects of pure equation M155 nutrition on shoot growth in wild-type plants, but not in atnrt2.1 mutants. This indicates that the protective action of 0.1 mm equation M156 against equation M157 toxicity is dependent on NRT2.1 expression.

DISCUSSION

A Novel Regulation of NRT2.1 Expression Involving NRT1.1-Mediated Repression by High NO3

Knowledge concerning the control of NRT2.1 expression by N is that this gene is under two main regulations, namely (1) induction by equation M158; and (2) repression by high N status of the whole plant (Filleur and Daniel-Vedele, 1999; Lejay et al., 1999; Zhuo et al., 1999; Forde, 2000; Gansel et al., 2001; Orsel et al., 2002; Nazoa et al., 2003; Okamoto et al., 2003). The molecular mechanisms underlying these regulations are unknown, but there is some consensus about the nature of the signal molecules involved. In particular, the equation M159 ion itself is believed to be the inducer (Crawford and Glass, 1998; Forde, 2000), and equation M160 and Gln are thought to be the main signal molecules involved in the feedback repression exerted by the N status of the plant (Lejay et al., 1999; Zhuo et al., 1999; Nazoa et al., 2003).

This model now appears to be incomplete because NRT2.1 overexpression in wild-type plants under low equation M161/high equation M162 availability (Figs. 144)) cannot be explained by the above mechanisms (i.e. induction by equation M163 and repression by equation M164). A striking illustration of this is the observation that transfer of the plants from 1 mm NH4NO3 to 5 mm equation M165 + 0.1 mm equation M166 led to 4-fold stimulation of NRT2.1 expression (Fig. 1B), although this corresponded to a 10-fold decrease in the concentration of the inducer (equation M167) and a 5-fold increase in the concentration of the repressor (equation M168). Furthermore, as already shown for up-regulation of NRT2.1 in NH4NO3-fed chl1 mutants (Muños et al., 2004), up-regulation of NRT2.1 by low equation M169 in the presence of equation M170 cannot be mistaken with relief of the feedback repression exerted by the overall N status of the plant: (1) plants subjected to low equation M171/high equation M172 availability have a high total N content of the tissues and thus have high N status; (2) root equation M173 influx is not up-regulated in these plants (Fig. 3B), whereas it is also under negative feedback control by the N status of the plant (Gazzarrini et al., 1999; Rawat et al., 1999; von Wirén et al., 2000); and (3) stimulation of NRT2.1 expression by low equation M174/high equation M175 availability is under purely local control (Fig. 2), whereas NRT2.1 regulation by the N status of the plant involves systemic signaling (Imsande and Touraine, 1994; Gansel et al., 2001; Forde, 2002).

Altogether, our data provide evidence that NRT2.1 expression is also modulated by a third important regulatory mechanism, triggering repression of this gene by high external equation M176 availability, which superimposes on repression exerted by equation M177 or Gln. Indeed, in the presence of either reduced N source, NRT2.1 expression in wild-type roots was consistently found to be primarily determined by external equation M178 concentration, with strong down-regulation as soon as this concentration exceeded the 0.2 to 0.5 mm range. Although surprising at first glance, the hypothesis that equation M179 may have opposite regulatory effects (induction and repression) is already well documented for its role in the control of lateral root growth (local stimulation of lateral root elongation and systemic repression of lateral root emergence; Zhang et al., 1999). Furthermore, down-regulation of root equation M180 uptake by equation M181 itself has already been postulated, in particular from barley (Hordeum vulgare) experiments where root equation M182 uptake was found to be negatively correlated with root equation M183 concentration, but only when root equation M184 concentration exceeded a certain threshold level (Siddiqi et al., 1989; Crawford and Glass, 1998). Although these physiological studies provided circumstantial evidence for repression of root equation M185 uptake by high equation M186, our results bring insight to these aspects because they highlight NRT2.1 and NRT3.1 as molecular targets of this regulation. Recently, NRT3.1 expression was also shown to be induced by equation M187 (Okamoto et al., 2006) and repressed by high N status of the plant (Remans et al., 2006). Thus, NRT3.1 appears to be, at least partially, controlled by the same regulatory network as NRT2.1, suggesting coordinated regulation of these two components of the HATS. Most importantly, our data also reveal specific involvement of NRT1.1 in triggering repression by high equation M188 (Figs. 4 and and5).5). Indeed, down-regulation of NRT2.1/NRT3.1 expression by high equation M189 availability in the presence of equation M190 is fully suppressed in the chl1-10 mutant (and not in a nrt1.2 mutant), whereas root equation M191 uptake is not reduced in chl1-10 compared to wild type (whereas it is reduced in atnrt1.2-1). This clearly invalidates one of our initial hypotheses that this phenotype of chl1 mutants is simply a compensatory response to a general defect in equation M192 acquisition and thus an indirect consequence of NRT1.1 mutation. Our proposal for the regulatory role of NRT1.1 in wild-type plants is that the increase in external equation M193 concentration results in an increasing activity of this transporter, which in turn generates an increasing repressive signal for NRT2.1 expression (Fig. 4A). Whether this indicates a direct signaling function for NRT1.1 (in analogy with the role of the equation M194 sensor recently proposed for NRT2.1) and calls for the specific involvement of one isoform of NRT1.1 (high or low affinity) are open questions that deserve further investigation.

To account for our observations, we propose a model for N regulation of NRT2.1 expression (Fig. 8). In addition to the positive regulation corresponding to the induction by equation M195, this model postulates dual negative regulation involving both feedback repression by reduced N metabolites and NRT1.1-mediated repression by high external equation M196. An important point is that the absence of NRT1.1-mediated repression (due to mutation of NRT1.1 or to low external equation M197 availability) overrides the negative feedback exerted by reduced N metabolites to yield a high NRT2.1 expression level even in the presence of ample equation M198 supply to the plant (e.g. 5 mm; see Fig. 1B). Conversely, there is also evidence that lack of negative feedback regulation by reduced N metabolites overrides the repressive effect of high external equation M199 availability. This is shown by the high NRT2.1 expression level in nitrate reductase-deficient plants supplied with equation M200 as a sole N source (Lejay et al., 1999; Zhuo et al., 1999). Taken together, these observations suggest that NRT2.1 expression is suppressed only when both negative regulations by reduced N metabolites and by high equation M201 are effective (as illustrated in Fig. 8). Whether this means that the two respective signaling pathways directly interfere at some common crucial node or, alternatively, that they are independently strong enough to overcome each other is not known. However, because NRT1.1 mutation prevents NRT2.1 repression by high equation M202, but does not alter its reinduction by equation M203 after a period of N starvation (Muños et al., 2004), it is concluded that these opposite actions of equation M204 most probably involve independent signaling pathways.

Figure 8.
Model for N regulation of NRT2.1 expression in Arabidopsis roots. The model postulates that, in addition to the induction by equation M336, NRT2.1 is also under dual control by feedback repression by reduced N metabolites and NRT1.1-mediated repression by high external ...

NRT1.1-Mediated Regulation of NRT2.1 Allows an Adaptive Response of the Plant to NH4+ Toxicity

A key issue concerning NRT1.1-mediated regulation of the HATS by high equation M205 is to determine what physiological role such a mechanism may play. In analogy with the well-accepted postulate that repression of root equation M206 (or equation M207) uptake systems by reduced N metabolites corresponds to a regulation by the N demand of the plant (Imsande and Touraine, 1994; vonWirén et al., 2000; Forde, 2002), we hypothesize that repression of NRT2.1 by high equation M208 corresponds to regulation by a equation M209 demand of the plant. Accordingly, relief of this NRT1.1-mediated repression due either to decreased equation M210 availability in the presence of equation M211, or to NRT1.1 mutation activated the equation M212 HATS but not the equation M213 uptake system (Fig. 3; Muños et al., 2004). This shows that root equation M214 uptake, and not total root N uptake, is the specific target of this mechanism. Clearly, what the plant perceives under these situations is a lack of equation M215 and not an overall nutritional N deficiency.

In this context, the significance of the model depicted in Figure 8 is that repression of NRT2.1 by reduced N metabolites in N-sufficient plants is allowed only when NRT1.1 is active in transporting equation M216. This warrants that a significant equation M217 uptake rate is always ensured in any situation, either by NRT1.1 or by NRT2.1, even when equation M218 accounts for only a minor fraction of the total N available in the external medium (Fig. 4B). One of the most obvious interests of such a mechanism is to protect the plant against equation M219 toxicity. It is known for decades that pure equation M220 nutrition is toxic for many plant species (Givan, 1979; Hageman, 1984; Salsac et al., 1987; Kronzucker et al., 2001). In particular, growth of herbaceous dicotyledons such as tomato (Lycopersicon esculentum), French bean (Phaseolus vulgaris), spinach (Spinacia oleracea), and here Arabidopsis, is generally strongly hampered by pure equation M221 nutrition, with a decrease in yield of up to 60% as compared with supply of equation M222 as the sole N source (Salsac et al., 1987). A general observation is, however, that equation M223 toxicity is fully prevented by supply of equation M224 as a N source together with equation M225 (Cox and Reisenauer, 1973; Kronzucker et al., 1999; Rahayu et al., 2005). Actually, highest growth rates are generally achieved with mixed equation M226 + equation M227 supplies (Cox and Reisenauer, 1973; Heberer and Below, 1989; Adriaanse and Human, 1993; Cao and Tibbitts, 1993).

Despite this firmly established role of equation M228 in preventing the detrimental effects of equation M229 nutrition, a strong paradox remained unresolved. On the one hand, equation M230 uptake by the plant was shown to ensure full protection against equation M231 toxicity and, on the other hand, equation M232 uptake systems were shown to be strongly repressed by the supply of high equation M233 concentration to the plant. To date, no mechanism was known to stimulate equation M234 uptake in the presence of potentially toxic concentrations of equation M235 in the external medium. We propose that the NRT1.1-mediated regulation of NRT2.1/NRT3.1 corresponds to such a mechanism because it relieves repression of the HATS under low equation M236/high equation M237 availability (Figs. 3A and and4).4). Furthermore, the phenotype of both atnrt2.1 mutants demonstrates that up-regulation of the HATS under this condition constitutes an essential adaptive response of the plant to avoid equation M238 toxicity (actually the only one documented at the molecular level; Figs. 6 and and7;7; Supplemental Figs. S2 and S3).

A surprising aspect of the HATS repression by high equation M239 is that it seems to rely on purely local signaling because only the portions of the root system subjected to low equation M240/high equation M241 availability react in up-regulating NRT2.1 expression (Fig. 2). Furthermore, high equation M242 supply on one portion of the root system does not prevent the adaptive response of NRT2.1 in other portions fed with excess equation M243 over equation M244 (see Fig. 3, lanes b and c). This suggests that the equation M245 demand governing NRT2.1 expression is not sensed at the whole-plant level and that the adaptive response of NRT2.1 aims at stimulating equation M246 uptake specifically in the root cells experiencing high external equation M247 availability. This is in full agreement with the results from split-root experiments on maize (Zea mays) and soybean (Glycine max), indicating that equation M248 plays its protective role against equation M249 toxicity only when it is locally supplied together with equation M250, and not when the two N sources are separately provided to only one half on the root sy stem (Schortemeyer et al., 1993; Saravitz et al., 1994).

Our data thus show that, besides its key role in ensuring the bulk of N acquisition by the plant in many various environmental conditions, NRT2.1 also plays a critical function in maintaining a healthy balance between equation M251 and equation M252 uptake. It is highlighted that, in this latter case, NRT2.1 activity is not required to supply an N source for amino acid synthesis, but to allow the plant to benefit from a specific role of equation M253 that equation M254 cannot fulfill. This illustrates very well why NRT2.1 cannot be regulated only by feedback repression by N metabolites because this regulation aims at adjusting N uptake to amino acid utilization and is not specific for equation M255 uptake systems (vonWirén et al., 2000). Interestingly, up-regulation of NRT2.1 expression by low equation M256 availability was also observed in the presence of Gln (Supplemental Fig. S1), suggesting that equation M257 demand signaling may be operative under other circumstances than those associated with equation M258 toxicity. It is thus tempting to postulate a more general role for this signaling in regulating equation M259 acquisition by the plant. Nitrate is not only a nutrient, but also a key signaling compound governing crucial aspects of plant metabolism and development (Crawford, 1995; Stitt, 1999). In particular, equation M260 regulates many genes related to N or C metabolism (Crawford, 1995; Stitt, 1999), triggers several adaptive responses of root and shoot growth (Forde, 2002; Walch-Liu et al., 2005), and modulates cytokinin signaling (Sakakibara, 2003). Thus, plants may have evolved specific regulatory mechanisms to tightly control these important signaling effects of equation M261. At the uptake level, this would require a regulatory mechanism specific for equation M262 transporters and independent from the feedback regulation by reduced N metabolites, which aims at ensuring efficient use of this ion (as well as of equation M263) as a nutrient. In this context, the model of Figure 8 corresponds to an elegant mechanism for integrating both requirements for equation M264 as a nutrient and as a signal in the regulation of root equation M265 uptake. Therefore, the question of whether NRT1.1-mediated regulation of NRT2.1 reported in this work has additional functions other than just protecting the plant from equation M266 toxicity deserves further investigation. In particular, given the role of NRT1.1 in regulating NRT2.1 expression, and the role of NRT2.1 in controlling lateral root initiation, it will be of interest to investigate whether putative equation M267 signaling mediated by NRT1.1 is also involved in modulating root branching as a function of external equation M268 availability.

MATERIALS AND METHODS

Plant Material and Treatments

The Arabidopsis (Arabidopsis thaliana) genotypes used in this study were the wild-type Ws and Columbia-0 ecotypes; the atnrt2.1-1 mutant in the Ws background (formerly atnrt2a), obtained from the collection of Institut National de la Recherche Agronomique, Versailles, and deleted for the NRT2.1 (At1g08090) and NRT2.2 (At1g08100) genes (Filleur et al., 2001); the atnrt2.1-2 mutant in the Columbia-0 background, obtained from the Salk Institute (SALK_035429), and carrying a T-DNA insertion in the first intron of NRT2.1 (these two mutants were renamed according to the nomenclature proposed by Little et al., 2005); and the atnrt1.2-1 mutant in the Ws background, obtained from the collection of the Institut National de la Recherche Agronomique, and carrying a T-DNA insertion in the third intron of NRT1.2. NRT1.2 mRNA was not detected by reverse transcription (RT)-PCR in the roots of this mutant (data not shown).

Plants were grown for 6 weeks in hydroponics under nonsterile conditions, as previously described by Lejay et al. (1999). The growth chamber was set with the following environmental conditions: 8-h light/16-h dark 22°C/20°C temperature, 250 μmol m−2 s−1 irradiance, and 70% hygrometry. Briefly, seeds were sown directly on sand contained by a cut 1.5-mL Eppendorf tube closed at the bottom by a stainless grid. Tubes were supported by PVC discs (six Eppendorf/disc) placed on a floating polystyrene raft (12 discs/raft). These systems were disposed on top of 10-L tanks filled with tap water for the first week, and then with nutrient solution for 4 to 5 additional weeks (during this period, nutrient solutions were renewed weekly). The basal nutrient solution common to all experiments included 1 mm KH2PO4, 1 mm MgSO4, 0.25 mm K2SO4, 0.25 mm CaCl2, 0.1 mm FeNa-EDTA, 50 μm KCl, 30 μm H3BO3, 5 μm MnSO4, 1 μm ZnSO4, 1 μm CuSO4, and 0.1 μm (NH4)6Mo7O24. For growth of the plants, 1 mm NH4NO3 was added to the basal medium as the N source. Depending on the experiments, 1 mm NH4NO3 was replaced as a N source by either KNO3 or NH4Cl, or various mixtures of these salts, as indicated in the text and figures. The pH of all solutions was adjusted to 5.8, and the solutions were renewed every other day during the experiments to prevent nutrient depletion. For experiments with media at low equation M269 concentration (0.1 mm), nutrient solutions were renewed daily, which allowed maintenance of the external equation M270 concentration above 0.06 to 0.07 mm. For treatments with Gln, 25 mg L−1 chloramphenicol and 50 mg L−1 penicillin were added to solutions to prevent microbial development. For time-course studies, various treatments were initiated at different times to allow harvest of all plants at the same time of the day (7–8 h into the light period) to prevent any diurnal effect on NRT2.1 expression (Lejay et al., 2003).

For split-root experiments, the protocol was reported previously (Gansel et al., 2001). At the age of 2 weeks, seedlings were cleared to leave only one plant per tube. After gentle separation of the root system into two approximately equal portions, 5-week-old plants were transferred to specific containers and allowed to adapt 3 d to split-root conditions, with the two parts of the root system supplied with the 1 mm NH4NO3 solution. The various treatments were then initiated at the end of this period.

For growth analysis, roots and shoot were separated after harvest and their fresh weight determined. Fresh weight data are the means of 10 to 18 replicates.

RNA Extraction and RNA Gel-Blot Analysis

RNA extraction was performed as previously described (Lobreaux et al., 1992) from eight to 12 plants per treatment (except for six plants in the split-root experiment). Ten micrograms of total RNA were then separated by electrophoresis on MOPS-formaldehyde agarose gel and blotted on nylon membrane (Hybond N+; Amersham-Pharmacia Biotech). Membranes were prehybridized for 2 h at 60°C in church buffer: 0.5 m NaHPO4, 1% bovine serum albumin, and 7% SDS (pH 7.2 with H3PO4). Hybridization was performed overnight at 60°C after addition of a randomly primed 32P-labeled cDNA probe in the hybridization buffer. Membranes were washed twice at root temperature for 2 min and twice at 60°C with 0.5× SSC, 0.1% SDS. The probe used in this study corresponds to the full-length of AtNRT2.1 cDNA (Filleur and Daniel-Vedele, 1999). A 25S rRNA probe was used to normalize quantifications achieved using a phosphor imager (BAS-5000; Fujifilm).

Quantitative RT-PCR

Ten to 15 μg of total RNA were digested by RQ-DNase (Promega). After phenol-chloroform purification and isopropanol precipitation, RNA was reverse transcribed to one-strand cDNA using Moloney murine leukemia virus reverse transcriptase (Promega) and dT(18) V primers, according to the manufacturer's protocol. Gene expression was determined by quantitative real-time PCR (LightCycler; Roche Diagnostics) using gene-specific primers: NRT2.1 (forward, 5′-aacaagggctaacgtggatg-3′ and reverse, 5′-ctgcttctcctgctcattcc-3′); NRT3.1 (forward, 5′-ggccatgaagttgcctatg-3′ and reverse, 5′-tcttggccttcctcttctca3-′); ACT2/8 (forward, 5′-ggtaacattgtgctcagrggtgg-3′ and reverse, 5′-aacgaccttaatcttcatgctgc-3′), and LightCycler FastStart DNA Master SYBR Green (Roche Diagnostics). Expression levels of tested genes were normalized to expression levels of the ACT2/8 genes (Charrier et al., 2002).

15NO3 and 15NH4+ Uptake

Influx of either 15equation M271 or 15equation M272 into the roots was assayed as described previously (Lejay et al., 1999) by 5-min labeling in basal nutrient medium (pH 5.8) supplemented with appropriate concentrations of K15NO3 or 15NH4Cl (atom % 15N excess: 99%). For specific determination of the activity of the HATS, 15equation M273 or 15equation M274 were at 0.2 mm in the labeling solution. Cumulative equation M275 uptake during long-term growth studies (10–12 d) was assayed by supplying the plants with nutrient solution containing 15equation M276 (atom % 15N excess: 1%) for the whole experimental period and by measuring total 15N accumulation in roots and shoots at the end of this period. Each influx or cumulative equation M277 uptake value is the mean of eight to 12 replicates.

The total N content and atomic percentage 15N abundance of the samples were determined by continuous-flow mass spectrometry, as described previously (Clarkson et al., 1996), using a Euro-EA Eurovector elemental analyzer coupled with an IsoPrime mass spectrometer (GV Instruments).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. Up-regulation of NRT2.1 in the presence of Gln.
  • Supplemental Figure S2. equation M278 toxicity in the atnrt2.1-2 mutant.
  • Supplemental Figure S3. equation M279 toxicity in the atnrt2.1-1 mutant.

Supplementary Material

[Supplemental Data]

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alain Gojon (gojon/at/ensam.inra.fr).

[W]The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.087510

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