Wang S et al. (2020), Functional analysis of the OsNPF4.5 nitrate tra...

XB-ART-57236

Proc Natl Acad Sci U S A 2020 Jul 14;11728:16649-16659. doi: 10.1073/pnas.2000926117.

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Functional analysis of the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants.

Wang S , Chen A , Xie K , Yang X , Luo Z , Chen J , Zeng D , Ren Y , Yang C , Wang L , Feng H , López-Arredondo DL , Herrera-Estrella LR , Xu G .

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Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus Rhizophagus irregularis remarkably promoted rice (Oryza sativa) growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO3- supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene OsNPF4.5 in rice roots, and its orthologs ZmNPF4.5 in Zea mays and SbNPF4.5 in Sorghum bicolor OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO3- transport activity when expressed in Xenopus laevis oocytes. Moreover, knockout of OsNPF4.5 resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO3- was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO3- acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.

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Species referenced: Xenopus laevis
Genes referenced: chl1 pam

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	Fig. 1. RNA sequencing analysis of the rice mycorrhizal and nonmycorrhizal roots. (A) Venn diagram showing the relationships between genes that show statistically significant differential expression in response to AM symbiosis in roots. The up-regulated genes are shown in red color, while the down-regulated genes are indicated in yellow color. The genes with no significant alteration in transcripts are shown in the intersection. (B) The 30 most significantly enriched pathways analyzed by KEGG algorithm. (C) A heat map of the up-regulated genes involved in nitrogen transport and metabolism, as well as several previously described AM–up-regulated genes that were shown as marker genes. (D) Quantitative RT-PCR analysis showed a more than 500-fold up-regulation of OsNPF4.5 and an 11-fold up-regulation of OsAMT3.1 in response to AM symbiosis. The AM-specific Pi transporter gene OsPT11 and H+-ATPase gene OsHA1 were used as control genes. The relative expression level of the assayed genes was normalized to a constitutive Actin gene. Values are the means ± SE of three biological replicates (n = 3). The asterisks indicate significant differences (P < 0.05; P < 0.01, **P < 0.001).
	Fig. 2. AM fungal colonization promotes rice growth and nitrate uptake. (A) A diagrammatic representation (not to scale) of the compartmented culture system used in the experiment. Two inoculated or mock-inoculated seedlings of WT or mutant plants were grown in the middle root/fungal compartment (RFC) and watered weekly with nutrient solution containing 2.5 mM NO3−. The hyphal compartments (HCs) aside were watered with nutrient solution containing an equal amount of 15NO3−. (B) Biomass of inoculated and mock-inoculated plants. (C) Assay of 15N content in both roots and shoots of inoculated and mock-inoculated plants. (D–G) N (D and E) and P (F and G) contents of inoculated and mock-inoculated plants. (H) The percentage of N and P transferred via the mycorrhizal pathway. Values are the means ± SE of five independent biological replicates (n = 5). The asterisks indicate significant differences (P < 0.05; P < 0.01, **P < 0.001).
	Fig. 3. Tissue-specific expression assay of OsNPF4.5 in response to AM symbiosis. (A) Transcripts of OsNPF4.5 in different tissues of mycorrhizal (AM) and nonmycorrhizal (NM) plants. (B–D) Time-course expression of OsNPF4.5 and OsPT11 (used as a control) in rice mycorrhizal roots. (D) Quantification of AM fungal colonization at different sampling time points. (E and F) Histochemical GUS staining of rice roots expressing pOsNPF4.5::GUS in the absence (E) and presence (F) of inoculation. (G) Magenta-GUS staining of the mycorrhizal roots. (H) Colocalization of GUS activity (indicated by the purple color from the overlay of the trypan blue and magenta-GUS stains). Red arrows indicate arbuscules. Blue arrows denote noncolonized cells in mycorrhizal roots. (Scale bars: 50 μm.)
	Fig. 4. Functional characterization of OsNPF4.5 in vitro and in vivo. (A and B) Results of nitrate-uptake assay in Xenopus oocytes injected with OsNPF4.5 and CHL1 cRNAs using 15N-nitrate at pH 5.5 (A) and pH 7.4 (B). CHL1 was used as a positive control. (C) Nitrate uptake kinetics of OsNPF4.5 in oocytes. OsNPF4.5 cRNA was injected into oocytes, which were incubated in the ND96 solution containing 0.25, 1, 2.5, 5, 10, 15, and 20 mM Na15NO3, respectively, for 2 h at pH 5.5. (D) Current–voltage curves of oocytes expressing OsNPF4.5. The I–V curves shown were recorded from OsNPF4.5- and H2O-injected oocytes, which were treated with 10 mM nitrate at pH 5.5. Values are means ± SE (n = 10 oocytes). (E and F) The 15N accumulation in roots of WT and OsNPF4.5-overexpressing plants under 15NO3− (E) or 15NH4+ (F) supply hydroponic conditions. In the uptake experiment, WT and OsNPF4.5-overexpressing transgenic lines, referred as OX lines, suffered from N deprivation for 4 d and then were resupplied with 15N-labled 2.5 mM NO3− or 2.5 mM NH4+ for 10 min. Values are means ± SE of five biological replicates (n = 5). The asterisks indicate significant differences (P < 0.05; P < 0.01, **P < 0.001).
	Fig. 5. Physiological analysis of the OsNPF4.5 loss function mutants. WT and three osnpf4.5 mutant lines generated by CRISPR-Cas9 were cultivated in a compartmented growth system containing a middle root/hyphal compartment (RHC) that was separated by 30-mm nylon meshes from two hyphal compartments (HCs). The RHC and HC were irrigated with 2.5 mM NO3− and 15NO3− weekly, respectively. The inoculated and mock-inoculated WT and osnpf4.5 plants were harvested for physiological analysis at 6 wpi. (A) Shoot biomass (dry weight), shoot N content (B and C), and 15N accumulation (D) of the WT and osnpf4.5 mutant plants inoculated with R. irregularis (AM) or mock-inoculated controls (NM). (E) The contribution of the symbiotic NO3− acquisition pathway to overall N uptake of WT and osnpf4.5 mutants. (F–L) The mycorrhizal colonization level (F) determined in hypha (labeled “H”), arbuscule (“A”), and vesicle (“V”) and arbuscule incidence and morphology in WT (G and K) and osnpf4.5 mutants (H–J and L). Values are means ± SE of five independent biological replicates (n = 5). Different letters and asterisks indicate significant differences (P < 0.05; *P < 0.01). (Scale bars: G–J, 50 μm; K and L, 25 μm.)
	Fig. 6. A model for N uptake, assimilation, and translocation in AM symbiosis. AM fungi can take up both NH4+ and NO3−, as well as organic N forms, such as amino acids (AAs) and small peptides (SPs), from soil solution via their extraradical mycelium (ERM). The NH4+ in fungal cytoplasm can be rapidly assimilated into amino acids, mainly arginine, via the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and translocated, probably coupled with Poly-P through the intraradical hyphae. After hydrolysis in the arbuscule, NH4+ is exported from the AM fungus to the periarbuscular space (PAS) and subsequently imported, probably in the form of NH3, into the root cell by the putative plant NH4+ transporters located on the periarbuscular membrane (PAM). The NO3− absorbed by extraradical mycelium can be directly translocated into intraradical hyphae and released into the interfacial apoplast. The import of NO3− into root cell is mediated by the PAM-localized NO3− transporters, such as OsNPF4.5. NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase; AMT, ammonium transporter, AAP, amino acid permease. Question marks and dotted lines indicate that the putative transporters or transport/metabolic processes have not yet been established.

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	Fig. 1. RNA sequencing analysis of the rice mycorrhizal and nonmycorrhizal roots. (A) Venn diagram showing the relationships between genes that show statistically significant differential expression in response to AM symbiosis in roots. The up-regulated genes are shown in red color, while the down-regulated genes are indicated in yellow color. The genes with no significant alteration in transcripts are shown in the intersection. (B) The 30 most significantly enriched pathways analyzed by KEGG algorithm. (C) A heat map of the up-regulated genes involved in nitrogen transport and metabolism, as well as several previously described AM–up-regulated genes that were shown as marker genes. (D) Quantitative RT-PCR analysis showed a more than 500-fold up-regulation of OsNPF4.5 and an 11-fold up-regulation of OsAMT3.1 in response to AM symbiosis. The AM-specific Pi transporter gene OsPT11 and H+-ATPase gene OsHA1 were used as control genes. The relative expression level of the assayed genes was normalized to a constitutive Actin gene. Values are the means ± SE of three biological replicates (n = 3). The asterisks indicate significant differences (P < 0.05; P < 0.01, **P < 0.001).
	Fig. 2. AM fungal colonization promotes rice growth and nitrate uptake. (A) A diagrammatic representation (not to scale) of the compartmented culture system used in the experiment. Two inoculated or mock-inoculated seedlings of WT or mutant plants were grown in the middle root/fungal compartment (RFC) and watered weekly with nutrient solution containing 2.5 mM NO3−. The hyphal compartments (HCs) aside were watered with nutrient solution containing an equal amount of 15NO3−. (B) Biomass of inoculated and mock-inoculated plants. (C) Assay of 15N content in both roots and shoots of inoculated and mock-inoculated plants. (D–G) N (D and E) and P (F and G) contents of inoculated and mock-inoculated plants. (H) The percentage of N and P transferred via the mycorrhizal pathway. Values are the means ± SE of five independent biological replicates (n = 5). The asterisks indicate significant differences (P < 0.05; P < 0.01, **P < 0.001).
	Fig. 3. Tissue-specific expression assay of OsNPF4.5 in response to AM symbiosis. (A) Transcripts of OsNPF4.5 in different tissues of mycorrhizal (AM) and nonmycorrhizal (NM) plants. (B–D) Time-course expression of OsNPF4.5 and OsPT11 (used as a control) in rice mycorrhizal roots. (D) Quantification of AM fungal colonization at different sampling time points. (E and F) Histochemical GUS staining of rice roots expressing pOsNPF4.5::GUS in the absence (E) and presence (F) of inoculation. (G) Magenta-GUS staining of the mycorrhizal roots. (H) Colocalization of GUS activity (indicated by the purple color from the overlay of the trypan blue and magenta-GUS stains). Red arrows indicate arbuscules. Blue arrows denote noncolonized cells in mycorrhizal roots. (Scale bars: 50 μm.)
	Fig. 4. Functional characterization of OsNPF4.5 in vitro and in vivo. (A and B) Results of nitrate-uptake assay in Xenopus oocytes injected with OsNPF4.5 and CHL1 cRNAs using 15N-nitrate at pH 5.5 (A) and pH 7.4 (B). CHL1 was used as a positive control. (C) Nitrate uptake kinetics of OsNPF4.5 in oocytes. OsNPF4.5 cRNA was injected into oocytes, which were incubated in the ND96 solution containing 0.25, 1, 2.5, 5, 10, 15, and 20 mM Na15NO3, respectively, for 2 h at pH 5.5. (D) Current–voltage curves of oocytes expressing OsNPF4.5. The I–V curves shown were recorded from OsNPF4.5- and H2O-injected oocytes, which were treated with 10 mM nitrate at pH 5.5. Values are means ± SE (n = 10 oocytes). (E and F) The 15N accumulation in roots of WT and OsNPF4.5-overexpressing plants under 15NO3− (E) or 15NH4+ (F) supply hydroponic conditions. In the uptake experiment, WT and OsNPF4.5-overexpressing transgenic lines, referred as OX lines, suffered from N deprivation for 4 d and then were resupplied with 15N-labled 2.5 mM NO3− or 2.5 mM NH4+ for 10 min. Values are means ± SE of five biological replicates (n = 5). The asterisks indicate significant differences (P < 0.05; P < 0.01, **P < 0.001).
	Fig. 5. Physiological analysis of the OsNPF4.5 loss function mutants. WT and three osnpf4.5 mutant lines generated by CRISPR-Cas9 were cultivated in a compartmented growth system containing a middle root/hyphal compartment (RHC) that was separated by 30-mm nylon meshes from two hyphal compartments (HCs). The RHC and HC were irrigated with 2.5 mM NO3− and 15NO3− weekly, respectively. The inoculated and mock-inoculated WT and osnpf4.5 plants were harvested for physiological analysis at 6 wpi. (A) Shoot biomass (dry weight), shoot N content (B and C), and 15N accumulation (D) of the WT and osnpf4.5 mutant plants inoculated with R. irregularis (AM) or mock-inoculated controls (NM). (E) The contribution of the symbiotic NO3− acquisition pathway to overall N uptake of WT and osnpf4.5 mutants. (F–L) The mycorrhizal colonization level (F) determined in hypha (labeled “H”), arbuscule (“A”), and vesicle (“V”) and arbuscule incidence and morphology in WT (G and K) and osnpf4.5 mutants (H–J and L). Values are means ± SE of five independent biological replicates (n = 5). Different letters and asterisks indicate significant differences (P < 0.05; *P < 0.01). (Scale bars: G–J, 50 μm; K and L, 25 μm.)
	Fig. 6. A model for N uptake, assimilation, and translocation in AM symbiosis. AM fungi can take up both NH4+ and NO3−, as well as organic N forms, such as amino acids (AAs) and small peptides (SPs), from soil solution via their extraradical mycelium (ERM). The NH4+ in fungal cytoplasm can be rapidly assimilated into amino acids, mainly arginine, via the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and translocated, probably coupled with Poly-P through the intraradical hyphae. After hydrolysis in the arbuscule, NH4+ is exported from the AM fungus to the periarbuscular space (PAS) and subsequently imported, probably in the form of NH3, into the root cell by the putative plant NH4+ transporters located on the periarbuscular membrane (PAM). The NO3− absorbed by extraradical mycelium can be directly translocated into intraradical hyphae and released into the interfacial apoplast. The import of NO3− into root cell is mediated by the PAM-localized NO3− transporters, such as OsNPF4.5. NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase; AMT, ammonium transporter, AAP, amino acid permease. Question marks and dotted lines indicate that the putative transporters or transport/metabolic processes have not yet been established.