Regulation of the high-affinity NO3- uptake system by NRT1.1-mediated NO3- demand signaling in Arabidopsis

Abstract

The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity NO(3)(-) transport system (HATS) that plays a crucial role in NO(3)(-) uptake by the plant. Although NRT2.1 was known to be induced by NO(3)(-) and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when NO(3)(-) concentration decreases to a low level (<0.5 mm) in media containing a high concentration of NH(4)(+) or Gln (>or=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 NO(3)(-) availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high NO(3)(-) is specifically mediated by the NRT1.1 NO(3)(-) transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up NO(3)(-) in the presence of a reduced N source. Under low NO(3)(-)/high NH(4)(+) 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 NH(4)(+) toxicity because NO(3)(-) taken up by the HATS in this situation prevents the detrimental effects of pure NH(4)(+) nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific NO(3)(-) demand of the plant in relation to the various specific roles that NO(3)(-) 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 NO(3)(-).

Figure 1.

Changes in NRT2.1 mRNA accumulation in response to various / 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. A, Time-course response in plants transferred to nutrient solution containing 1 mm plus 0.1 mm . B, Effect of transfer for 4 d to media containing various combinations of and concentrations. Transcript accumulation was monitored in roots both by northern-blot analysis (gel-blot images) and real-time RT-PCR (bar graphs) on the same samples. A 25S rRNA signal is presented as a loading control for northern blots. Quantification by real-time RT-PCR was normalized using actin genes as controls. Errors bars represent se (n = 3). Different letters on top of the bars indicate statistically significant differences (P < 0.05; t test).

Figure 2.

Effect of local changes in the / 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 separation of their root system into two approximately equal sides, plants were transferred to specific containers and left undisturbed during 3 d in the 1 mm NH₄NO₃ solution before application of localized supplies with different / mixtures for 4 d, as indicated in the figure. B, NRT2.1 mRNA accumulation. Transcript accumulation was monitored in various sides of the split-root systems both by northern-blot analysis (gel-blot images) and real-time RT-PCR (bar graphs) on the same samples. The lane labels relate to the various localized treatments as shown in A. A 25S rRNA signal is presented as a loading control for northern blots. Quantification by real-time RT-PCR was normalized using actin genes as controls. Errors bars represent se (n = 3). Different letters on top of the bars indicate statistically significant differences (P < 0.05; t test).

Figure 3.

Effect of various / 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 media containing the same concentration (1 mm), but various concentrations of (0.1, 1, or 10 mm). A, NRT2.1 mRNA accumulation and root ¹⁵ influx. Transcript accumulation was monitored in roots both by northern-blot analysis (gel-blot images) and real-time RT-PCR (bar graphs) on the same samples. A 25S rRNA signal is presented as a loading control for northern blots. Quantification by real-time RT-PCR was normalized using actin genes as controls. Errors bars represent se (n = 3). Root ¹⁵ influx was assayed by 5-min labeling at 0.2 mm external ¹⁵ concentration to specifically determine activity of the HATS. Each value is the mean of eight to 12 replicates ± se. B, Root ¹⁵ influx. Root ¹⁵ influx was assayed by 5-min labeling at 1 mm external ¹⁵ concentration to determine the uptake activity at the same concentration as for growth and treatment of the plants. Each value is the mean of eight to 12 replicates ± se. N.D., Not determined. Different letters on top of the bars indicate statistically significant differences (P < 0.05; t test).

Figure 4.

Relationship between NRT2.1 mRNA accumulation and root influx as a function of concentration in the presence of 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 containing 1 mm NH₄NO₃, before transfer for 4 d to the nutrient solutions containing 1 mm and at various concentrations, as indicated in the figure. A, NRT2.1 mRNA accumulation. Transcript accumulation was monitored in roots both by northern-blot analysis (gel-blot images) and real-time RT-PCR (graph) on the same samples. A 25S rRNA signal is presented as a loading control for northern blots. Quantification by real-time RT-PCR was normalized using actin genes as controls. Errors bars represent se (n = 3). Differences between chl1-10 and the other genotypes are statistically significant at **P < 0.01 and ***P < 0.001 (t test). B, Root ¹⁵ influx. Root ¹⁵ influx was assayed by 5-min labeling at the external ¹⁵ concentration corresponding to that supplied to the plants during the 4-d treatment. Each value is the mean of six to 12 replicates ± se. C, Plot of NRT2.1 mRNA accumulation (A) against root ¹⁵ influx (B).

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 concentration in the presence of 1 mm in the external medium. The experiment is the same as in Figure 5. B, Correlation between changes in NRT2.1 and NRT3.1 mRNA accumulation. All RNA samples used to determine changes in NRT2.1 expression presented in Figures 1 to 4 and Supplemental Figure S1 were analyzed for NRT3.1 mRNA accumulation. The figure presents the relative changes in transcript level for both NRT2.1 and NRT3.1 normalized to the control (Ws plants left on 1 mm NO₃NH₄) of each experiment. Gray squares, Experiment of Figure 1; white diamonds, experiment of Figure 2; gray triangles, experiment of Figure 3; white hexagons, experiment of Supplemental Figure S1; black circles, experiment of Figure 4.

Figure 6.

Effect of NRT2.1 mutation on the protective effect of against 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 alone or in combination with 0.1 mm ).

Figure 7.

Shoot growth and root uptake of Ws and atnrt2.1-1 mutant plants supplied with 1 mm with or without 0.1 mm . Plants were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mm NH₄NO₃ before transfer for 12 d to media containing either 1 mm alone or 1 mm plus 0.1 mm ¹⁵. A, Shoot fresh weight. Values are the means of 10 to 18 replicates ± se. B, Cumulative ¹⁵ uptake from the 1 mm plus 0.1 mm ¹⁵ solution during the 12-d period determined by total ¹⁵N analysis in both root and shoots at the end of the period. Values are the mean of 10 to 12 replicates ± se. Differences between wild type and mutant are statistically significant at **P < 0.01, ***P < 0.001 (t test). Ns, Not significant (P > 0.05).

Figure 8.

Model for N regulation of NRT2.1 expression in Arabidopsis roots. The model postulates that, in addition to the induction by , NRT2.1 is also under dual control by feedback repression by reduced N metabolites and NRT1.1-mediated repression by high external availability, which are both required to suppress NRT2.1 expression.