The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches

Abstract

Localized proliferation of lateral roots in NO(3)(-)-rich patches is a striking example of the nutrient-induced plasticity of root development. In Arabidopsis, NO(3)(-) stimulation of lateral root elongation is apparently under the control of a NO(3)(-)-signaling pathway involving the ANR1 transcription factor. ANR1 is thought to transduce the NO(3)(-) signal internally, but the upstream NO(3)(-) sensing system is unknown. Here, we show that mutants of the NRT1.1 nitrate transporter display a strongly decreased root colonization of NO(3)(-)-rich patches, resulting from reduced lateral root elongation. This phenotype is not due to lower specific NO(3)(-) uptake activity in the mutants and is not suppressed when the NO(3)(-)-rich patch is supplemented with an alternative N source but is associated with dramatically decreased ANR1 expression. These results show that NRT1.1 promotes localized root proliferation independently of any nutritional effect and indicate a role in the ANR1-dependent NO(3)(-) signaling pathway, either as a NO(3)(-) sensor or as a facilitator of NO(3)(-) influx into NO(3)(-)-sensing cells. Consistent with this model, the NRT1.1 and ANR1 promoters both directed reporter gene expression in root primordia and root tips. The inability of NRT1.1-deficient mutants to promote increased lateral root proliferation in the NO(3)(-)-rich zone impairs the efficient acquisition of NO(3)(-) and leads to slower plant growth. We conclude that NRT1.1, which is localized at the forefront of soil exploration by the roots, is a key component of the NO(3)(-)-sensing system that enables the plant to detect and exploit NO(3)(-)-rich soil patches.

Fig. 1.

Mutation of NRT1.1 alters LR elongation in a nitrate-rich patch. (A) Response of the root system architecture to a localized high NO₃⁻ supply (HN; 10 mM) in wild-type (Ws) and NRT1.1 mutant (chl1-10) of Arabidopsis. LN, low NO₃⁻ medium (0.05 mM). DAT, day after transfer (Scale bars: 1 cm.) (B and C) Stimulation of LR growth in the NO₃⁻-rich patch with total (B) and mean (C) length of second-order LRs on the HN side. DAT, day after transfer. Errors bars represent SEM (n = 10–14).

Fig. 2.

Mutation of NRT1.1 does not alter specific nitrate uptake activity but reduces growth of plants subjected to localized nitrate supply. (A) Ratio between root dry biomass in HN (10 mM) and LN (0.05 mM) patches in two NRT1.1 mutant alleles (chl1-10 and chl1-5) and related wild-types (Ws and Col) after 12 d of growth. (B and C) Cumulative ¹⁵NO₃⁻ uptake by the roots on ¹⁵NO₃⁻-labeled HN patch expressed on the total plant dry weight basis either 1 d (B) or 12 d (C) after transfer to HN/LN medium. (D) Total plant dry biomass 12 d after transfer to HN/LN medium. DAT, day after transfer. Errors bars represent SEM (n = 6–14). Means for WT and chl1 mutants significantly different (Student t test) at: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. n.s., not significant (P > 0.1).

Fig. 3.

The root phenotype of chl1 mutants is due to altered sensing of nitrate. (A) Response of the root system architecture to a localized high NO₃⁻ availability in Arabidopsis plants with a root system pruned to two first-order LRs, placed for 5 d either on high (10 mM, HN) or low (0.05 mM, LN) NO₃⁻ medium, respectively. DAT, day after transfer to HN/LN medium (Scale bars: 1 cm.). (B–E) Effect of N composition of HN and LN media on the ratio at DAT5 between total second-order LR length in HN and LN sides of the root system. Means for WT and chl1 mutants significantly different (Student's t test) at: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. (F) Time course of second-order LR growth in plants subjected to heterogeneous NO₃⁻ supply (HN/LN) with HN at either 0.5 or 10 mM and LN at 0 or 0.05 mM, respectively. Errors bars represent SEM (n = 8–15).

Fig. 4.

Spatial localization of NRT1.1 and ANR1 expression. (A–C and G–I) Histochemical localization of GUS activity in pNRT1.1::GUS plants. (D–F and J–K) Histochemical localization of GUS activity in pANR1::GUS plants. GUS activity was visualized in LRs (A–F), emerging LR primordia (G and J), unemerged LR primordia (H and K), and primary root apex (I and L). Plants were grown on 0.5 mM NO₃⁻ (B, C, and E–L) or on 0.1 mM NO₃⁻ plus 0.5 mM gln (A and D) as the N source. (Scale bars: A, B, D, and E 150 μm; C and F–L, 50 μm.)

Fig. 5.

Effect of NRT1.1 mutation on ANR1 expression. (A) Relative ANR1 mRNA levels in the apical 10–15 mm of primary and LRs of wild-type and chl1 mutant plants, grown on homogenous medium containing either 0.5 or 10 mM NO₃⁻ as the N source. The values are the means from two replicate experiments. (B) Relative ANR1 mRNA levels in the apical 10–15 mm of first order or in second-order LRs of wild-type and chl1 mutant plants transferred for 3 d to heterogenous HN/LN medium (10/0.05 mM NO₃⁻). The data presented are those obtained for the roots the in HN side.