OsNLP3 and OsPHR2 orchestrate direct and mycorrhizal pathways for nitrate uptake by regulating NAR2.1-NRT2s complexes in rice

Abstract

Nitrogen (N) is the most important essential nutrient required by plants. Most land plants have evolved two N uptake pathways, a direct root pathway and a symbiotic pathway, via association with arbuscular mycorrhizal (AM) fungi. However, the interaction between the two pathways is ambiguous. Here, we report that OsNAR2.1-OsNRT2s, the nitrate (NO₃^-) transporter complexes with crucial roles in direct NO₃^- uptake, are also recruited for symbiotic NO₃^- uptake. OsNAR2.1 and OsNRT2.1/2.2 are coregulated by NIN-like protein 3 (OsNLP3), a key regulator in NO₃^- signaling, and OsPHR2, a major regulator of phosphate starvation responses. More importantly, AM symbiosis induces expression of OsNAR2.1-OsNRT2s, OsNLP3, and OsSPX4, encoding an intracellular Pi sensor, in arbuscular-containing cells, but weakens their expression in the epidermis. OsNAR2.1 and OsNLP3 can activate both mycorrhizal NO₃^- uptake and mycorrhization efficiency. Overall, we demonstrate that OsNLP3 and OsPHR2 orchestrate the direct and mycorrhizal NO₃^- uptake pathways by regulating the NAR2.1-NRT2s complexes in rice.

Keywords: NLP transcriptional factor; OsNAR2.1; PHR2; mycorrhizal pathway; nitrate uptake.

Fig. 1.

AM colonization promotes nitrate uptake and assimilation. (A) A diagrammatic representation (not to scale) of the compartmented culture system used in the experiment. The compartmented culture system contained a middle RFC and two HCs. Each HC was separated from the RFC by a 30-μm nylon mesh (green) and two pieces of iron gauze (black) with a 0.5-cm air gap. The nylon mesh fixed between the two iron gauzes permitted access to hyphae but prevented access of roots from the RFC to the HC. (B) Assay of ¹⁵N concentrations in roots and shoots of wild-type rice plants inoculated (AM) or mock-inoculated (NM) with Rhizophagus irregularis. Plants were grown in the RFC and fertilized weekly with 250 mL nutrient solution containing 2.5 mM NO₃⁻ or 2.5 mM NH₄⁺ as the N source for 8 wk. An equal amount of nutrient solution containing 2.5 mM ¹⁵NO₃⁻ or ¹⁵NH₄⁺ was supplied to the HCs side. (C) The contributions of N transferred via the mycorrhizal pathway under 2.5 mM NO₃⁻ and NH₄⁺ supply conditions, respectively. (D) Relative transcript levels of OsNR2 and OsNiR1 in rice mycorrhizal and nonmycorrhizal roots. (E–J) Histochemical GUS staining of rice roots expressing proOsNR2/proOsNiR1::GUS in the absence (E and H) and presence (F, G, I, and J) of R. irregularis inoculation. (G and J) WGA488 staining was used to observe the hyphal structures in the same root segments (F and I). Values are the means ± SE of 3 (D) or 4 (B and C) biological replicates. The asterisks indicate significant differences by two-tailed Student’s t test analysis (*P <0.05; **P < 0.01, ***P < 0.001). Red arrows indicate arbuscules. (Scale bars, 50 μm.)

Fig. 2.

OsNAR2.1 and OsNRT2s were induced in arbuscule-containing cells of rice mycorrhizal roots. (A–C) Relative transcript levels of OsNAR2 and OsNRT2 family genes in rice mycorrhizal (AM) and nonmycorrhizal (NM) roots treated with different N concentrations and forms for 6 wk, (A) 0.25 mM NO₃⁻, (B) 2.5 mM NO₃⁻ and (C) 1.25 mM NH₄NO₃. OsActin and OsPT11 were used as the reference and AM marker genes, respectively. Values are the means ± SE of three biological replicates. The asterisks indicate significant differences by two-tailed Student’s t test analysis (*P < 0.05; **P < 0.01, ***P < 0.001). (D) Spatial expression localizations of OsNAR2.1, OsNRT2.1, OsNRT2.2, and OsNRT2.3 in rice NM and mycorrhizal (AM) roots shown by promoter–GUS assays. Images in the sixth column represent the overlapped staining of WGA488 and GUS activity regarding the same root segments displayed in the fifth column. Images in the second, fourth, and seventh columns indicate cross sections of corresponding roots. The red arrows indicate the GUS staining in the arbuscule-containing cells and the black arrows show root hairs. (Scale bars, 50 μm.)

Fig. 3.

Physiological analysis of the OsNAR2.1 loss-of-function mutants. WT and osnar2.1 mutants were cultivated in a compartmented growth system containing a middle RFC separated by 30-mm nylon meshes from two HCs. The RFC and HC were irrigated with 0.25 mM or 2.5 mM NO₃⁻ and equivalent ¹⁵NO₃⁻ weekly, respectively. WT and osnar2.1 plants inoculated or mock-inoculated with R. irregularis were harvested for physiological analysis at 8 wpi. (A–H) ¹⁵N contents in shoots and roots (A and B), total shoot and root N contents (C and D), root length colonization levels (E and F), and relative transcript levels of AM marker genes (G and H) of WT and osnar2.1 plants supplied with 0.25 mM (A, C, E, and G) and 2.5 mM (B, D, F, and H) NO₃⁻, respectively. (I) WT and osnar2.1 mycorrhizal roots stained with WGA488, a dye that can stain the fungal structures with green fluorescence. Data represent means ± SE of 6 (A–D), 10 (E and F), and 4 (G and H) biological replicates. Different letters (one-way ANOVA Duncan’s test, P < 0.05) and asterisks indicate significant differences (two-tailed Student’s t test, *P < 0.05; **P < 0.01, ***P < 0.001). (Scale bars, 50 μm.)

Fig. 4.

OsNLP3 regulates both direct and mycorrhizal NO₃⁻ uptake pathways. (A–C) Spatial expression patterns of OsNLP3 in NM and mycorrhizal (AM) rice roots were revealed using the promoter–GUS assay (A and B) and WGA488 staining (C). (D–I) Physiological analyses of WT and osnlp3 mutants under compartmented growth conditions. (D and E) Root length colonization levels of WT plants and osnlp3 mutants at 4 and 8 wpi, respectively. (F) WT and osnar2.1 mycorrhizal roots stained with WGA488. (G) Arbuscule morphology of WT and osnlp3 mutants. (H and I) Total N contents (H) and ¹⁵N concentrations (I) in roots and shoots of WT and osnlp3 mutants. (J) Relative transcript levels of OsNAR2.1 in mycorrhizal and NM roots of WT and osnlp3 mutants at 2.5 mM NO₃⁻ supply conditions. (K–M) OsNLP3 interacts with OsNAR2.1 promoter in vitro and in vivo. (K) The locations of the NRE-like elements (green box) and the primers (black arrows) used for ChIP-qPCR analysis in the OsNAR2.1 promoter. The amplified promoter regions were indicated with purple boxes. (L) ChIP-qPCR analysis of the binding of OsNLP3 to NRE-like elements in OsNAR2.1 promoter. (M) OsNLP3 directly binds to NRE-like elements in OsNAR2.1 promoter by EMSAs. Plants used in all the experiments were cultivated under 2.5 mM NO₃⁻ condition. Data are means ± SE of 3 (L), 6 (D), 8 (E), and 4 (H–J) biological repeats. Different letters (one-way ANOVA Duncan’s test, P < 0.05) and asterisks indicate significant differences (two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001). (Scale bars, 50 μm.)

Fig. 5.

OsPHR2 regulates mycorrhizal NO₃⁻ uptake via directly modulating OsNAR2.1 and OsNRT2.1/2.2. (A) EMSAs of the bindings of OsPHR2 to the P1BS elements (GNATATNC) in the promoters of OsNAR2.1, OsNRT2.1, and OsNRT2.2. mP1BS (TNCGCGNA) indicates targeted mutation of the P1BS motif in the corresponding promoter fragments. (B) Transactivation assays of the promoters of OsNAR2.1, OsNRT2.1, and OsNRT2.2 by OsPHR2 in rice protoplasts with the luciferase reporter assays. EV (pGreen800-LUC vector), EV1 (pTCK303 vector). (C and D) Physiological analyses of WT and osphr2 mutants under compartmented growth conditions. (C) Shoot N contents in WT and osphr2 plants. (D) ¹⁵N concentrations in roots and shoots of WT and osphr2 plants. (E and F) Luciferase activity assay in N. benthamiana leaves transiently expressing Ubi-OsPHR2 or Ubi-OsNLP3, 35S-OsSPX4, and luciferase reporter driven by OsNAR2.1 promoter. Luciferase intensity was imaged at 36 h after infiltration (E) and quantified by Image-J (F). EV (pGreen800-LUC vector). EV2 (pCAMBIA1305 vector). Data are means ± SE of 3 (B) and 4 (C–F) biological replicates. Different letters (one-way ANOVA Duncan’s test, P < 0.05) and asterisks indicate significant differences (two-tailed Student’s t test, *P < 0.05).

Fig. 6.

OsNLP3 and OsPHR2 cotarget genes involved in NO₃⁻ transport and assimilation and AM symbiosis. (A) Distributions of OsNLP3 enriched peaks in rice genomic regions. (B) The enriched consensus sequences identified by Multiple Em for Motif Elicitation (MEME) motif analysis. (C) The Venn diagram showing the overlap of the OsNLP3 and PHR2 DAP-seq targets with RNASeq DEGs with enhanced expression in rice mycorrhizal roots. (D) A heat map of fold enrichment of the selected OsNLP3 DAP-seq targets involved in NO₃⁻ uptake and assimilation and AM symbiosis coupled with fold changes of RNAseq-based transcript accumulation in NM and AM roots of WT plants. (E) Transactivation assays of OsPHR2 and OsNLP3 on the promoters of OsNiR1, OsD17, OsDMI3, and OsRLI1 in rice protoplasts by dual-luciferase reporter system. Data are means ± SE of 3 (E) biological replicates and asterisks indicate significant differences (two-tailed Student’s t test, **P < 0.01, ***P < 0.001).

Fig. 7.

A proposed model for the coordinated regulation of NO₃⁻ uptake via direct and mycorrhizal pathways. NO₃⁻ is the dominant inorganic N form in aerobic soils. AM plants possess two pathways for NO₃⁻ uptake from surrounding soil, namely, (A) the direct pathway (DP) and (B) the mycorrhizal/symbiotic pathway (MP). In the direct pathway (A and C), NO₃⁻ in the rhizospheric soil is directly taken up by plant roots with the NO₃⁻ transporters, such as NAR2.1/NRT2s, located on the root hairs and root epidermis. In the mycorrhizal pathway (B and D), NO₃⁻ can be captured by the extensive ERM far away from the rhizosphere (B). A part of the NO₃⁻ acquired by AM fungi may be assimilated in the mycelium and used by the fungi for their own metabolism, and the remaining NO₃⁻ can be directly delivered into the interfacial apoplast, and subsequently imported into root cortical cells by plant NO₃⁻ transporters located at the PAM, including the NAR2.1/NRT2s, and OsNPF4.5 (D). The direct and mycorrhizal NO₃⁻ uptake pathways are coordinately regulated by a common regulatory network involving OsNLP3, OsPHR2, and OsSPX4, all of which showed a shift of expression localization from the epidermis and cortex to arbuscule-containing cells upon formation of AM symbiosis. OsNLP3 and OsPHR2 can directly regulate NAR2.1/NRT2s by binding to the NRE and P1BS motifs in their promoters, while OsSPX4 can interact with both OsNLP3 and OsPHR2 in the cytosol, thereby hindering their translocation into the nucleus. High NO₃⁻ accumulation in colonized root cells may trigger the degradation of OsSPX4 via the 26S proteasome pathway, resulting in the release of OsPHR2 and OsNLP3 to activate the symbiotic Pi-NO₃⁻ uptake and assimilation as well as the AM symbiosis regulatory pathways (E).