NIN-like protein 7 transcription factor is a plant nitrate sensor

Abstract

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants' primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.

Fig. 1.. Combinatorial NLP transcription factors are central to primary nitrate responses and developmental programs.

(A) NLPs regulate both common and distinct target genes. Hierarchical clustering of RNA-seq analysis defines putative target genes of NLP1–9 by transient expression in leaf cells. NLP protein expression was determined by immunoblot analyses. (B) Heatmap of nitrate-responsive core genes and unique target genes activated by NLP1–9. NA: nitrate assimilation; NT: nitrate transporter; OPP: oxidative pentose phosphate; TF: Transcription factor; IAA: IAA biosynthesis; PEP: peptide hormone; RC: root cap; CC: cell cycle; NM: nitrogen metabolism; CYT: cytokinin biosynthesis and signaling; SUC: sucrose invertase; AT: auxin transport; DNA: DNA synthesis; SAM: shoot apical meristem; RAM: root apical meristem. (C) The nlp2,4,5,6,7,8,9 mutant abolishes shoot growth in soil. (D) The nlp2,4,5,6,7,8,9 mutant displays nitrate-specific reduction in shoot and biomass. Error bars, s.d., n = 6. (E) Primary nitrate-responsive transcriptome is abolished in nlp2,4,5,6,7,8,9. (F) Nitrate uptake in nlp2,4,5,6,7,8,9. Error bars, s.d., n = 3. Scale bar, 1 cm.

Fig. 2.. Nitrate derepresses NLP7.

(A) Reporters and effectors for transient assays in leaf cells. The nitrate-responsive reporter (4xNRE-min-LUC) contains four copies of nitrate responsive element (NRE) and the 35S minimal promoter (min) fused to firefly luciferase gene (LUC) and NOS terminator (NOS). The 35S Cauliflower mosaic virus promoter controls the Renilla luciferase gene (pCAMV-RLUC) as the internal control. The effector expression is controlled by a constitutive HBT promoter (19). Numbering are the amino acid positions. (B) Analyses of NLP-NIN chimeras reveal de-repression of NLP7 by nitrate. Transactivation assays of NRE-LUC was carried out in nlp2,4,5,6,7,8,9 leaf cells by expressing effectors for 4 h before induction (0.5 mM KNO₃ for 2 h). Luciferase activity was normalized to pCAMV-RLUC activity. Error bars, s.d., n = 5 biological replicates. Effector protein expression was determined by immunoblot analyses. (C) Reporters and effector for transient assays in 293T human cells. The nitrate-responsive Human-4xNRE-min-LUC reporter contains the SV40 terminator. The herpes simplex virus (HSV) promoter driven RLUC (pHSV-RLUC) is an internal control. The MYC-NLP7-mGFP gene is controlled by a human cytomegalovirus (CMV) promoter with the β-globin gene terminator. (D) NLP7 activates Human-4xNRE-min-LUC in response to nitrate in heterologous 293T human cells. Luciferase activity was normalized to pHSV-RLUC activity (10 mM KNO₃ for 2 h). Error bars, s.d., n = 3 biological replicates.

Fig. 3.. Nitrate directly binds to NLP7.

(A) NO₃⁻ binding to full-length NLP7. Microscale thermophoresis (MST). Dissociation constant, K_d = 52 ± 20 μM. Error bar, s.d., n = 3. (B) NO₃⁻ binding to N-NLP7. K_d = 86 ± 38 μM. Error bar, s.d., n = 3. (C) Analysis of NO₃⁻-N-NLP7 interaction. Surface plasmon resonance (SPR). The result is a representative of three independent experiments. The K_d is the average of three independent experiments. (D) ClO₃⁻ does not bind to full-length NLP7. Error bars, s.d., n = 3. (E) ClO₃⁻ does not bind to N-NLP7. Error bars, s.d., n = 3. (F) SPR analysis of ClO₃⁻ -N-NLP7 interaction. The result is a representative of three independent experiments.

Fig. 4.. The genetically encoded biosensor detects intracellular nitrate in transgenic shoots and roots.

(A) A schematic representation of domain structure and a model of the nitrate biosensor. Nitrate triggers a conformational change of split mCitrine-NLP7 nitrate biosensor (sCiNiS) and reconstitutes mCitrine to emit fluorescent signals. The predicted nuclear localization signal (₆₃₀RRKKK₆₃₈) of NLP7 was mutated to AAAAA to avoid competition with the nitrate induced endogenous NLP7 in the nucleus. (B) Imaging the cytoplasmic nitrate by sCiNiS in mesophyll cells of cotyledons. Dashed white line, the imaging site. Images are representative of 10 cotyledons. Blue scale bar, 1 mm. White scale bar, 30 μm. (C) Imaging the cytoplasmic nitrate by sCiNiS in in root tips. Dashed red line, the imaging site. Images are representative of 10 root tips, KCl or KNO₃ (10 mM). (F-F₀)/F₀, the relative fluorescence intensity. Error bars, s.d., n = 10.

Fig. 5.. The NLP7 sensor domain resembles NreA with conserved residues for nitrate perception and signaling.

(A) The nitrate binding domain of the bacterial nitrate sensor NreA shares homology to a region of NLP7. The homologies are in red. Numbering refers to amino acid positions. (B) Sequence alignment of the nitrate binding domain of NreA and NLP7. Black box: conserved residue. Gray box: semi-conserved residue. Three essential nitrate binding residues are outlined in red. Asterisk, the conserved residues in the nitrate-binding pocket of the NreA. (C) Comparison of the crystal structure of the NreA nitrate-binding domain and the predicted structure of NLP7. Red stick: Nitrate. Pink: NreA (I59, L61, Y95) or NLP7(H404, L406, Y436). (D) Nitrate-binding mutant screens by transient assays in leaf cells. Transactivation of 4xNRE-min-LUC by NLP7 or nitrate-binding mutants was analyzed in nlp2,4,5,6,7,8,9 leaf protoplasts (0.5 mM KNO₃, 2 h). Error bars, s.d., n = 5 biological replicates. (E) The key nitrate binding residues are conserved in NLPs from major crops. Critical nitrate-binding residues are outlined in red. (F) Functional conservation of NLP7 and rice OsNLP3 for nitrate perception and transcription activation in heterologous 293T human cells. The nitrate-binding residues of NLP7(H404, L406, Y436) or OsNLP3(H392, L394, Y424) are essential for nitrate activation of human-4xNRE-min-LUC. Error bars, s.d., n = 3. (G)(H)(I) Mutant full-length NLP7 (HLY/AAA) and N-NLP7(HYL/AAA) abolish nitrate binding. Error bars, s.d., n = 3. The result is a representative of three independent experiments in SPR assays. (J) pNLP7-NLP7-GFP but not pNLP7-NLP7(HLY/AAA)-GFP complements the nlp7–1 mutant. Scale bars, 1 cm.