OsNRT1.1B‐OsCNGC14/16‐Ca²⁺‐OsNLP3 Pathway: Phosphorylation‐Mediated Maintenance of Nitrogen Homeostasis

Abstract

Nitrate, a crucial nutrient and signaling molecule, is extensively studied across plants. While the NRT1.1‐NLP‐centered pathway dominates nitrate signaling in Arabidopsis and rice, however, whether there is functional interaction or co‐regulation between the primary nitrate response (PNR) and long‐term nitrogen utilization remains unclear. Here, a novel nitrate signaling pathway is identified in rice that works alongside the established ubiquitination‐mediated OsNRT1.1B‐OsSPX4‐OsNLP3 cascade. It is demonstrated that OsCNGC14, OsCNGC16, and OsNRT1.1B form a plasma membrane‐localized complex in root tips, mediating nitrate‐triggered Ca²⁺ influx. The absence of either OsCNGC14 or OsCNGC16 abolished Ca²⁺ signaling and suppressed PNR. The OsNRT1.1B‐OsCNGC14/16 complex activates Ca²⁺‐dependent phosphorylation of OsNLP3 at Ser193, which accelerates its nuclear translocation and transcriptional activation of nitrate‐responsive genes. This phosphorylation enhances both short‐term PNR and long‐term nitrogenutilization. This findings reveal a dual regulatory network in rice: the Ca²⁺‐OsNLP3 pathway rapidly amplifies nitrate signals, while the ubiquitination‐mediated OsSPX4 degradation ensures sustained nitrogen homeostasis.

OsNRT1.1B forms a membrane complex with OsCNGC14/16 that mediates nitrate‐triggered calcium influx. This calcium signal phosphorylates OsNLP3 at Ser193, accelerating its nuclear translocation and activating nitrogen‐responsive genes. The calcium‐dependent pathway complements the established ubiquitination‐mediated pathway, dynamically regulating nitrogen homeostasis and enhancing nitrogen utilization efficiency in rice.

1. Introduction

Nitrogen (N) availability profoundly impacts plant growth, development, and agricultural productivity,^{[ 1} , ² , ^{3 ]} and consequently shapes global primary production.^{[ 4 ]} However, excessive dependence on synthetic fertilizers poses economic and environmental challenges.^{[ 5} , ⁶ , ^{7 ]} Nitrate, a primary N source for plants, serves both as a nutritional element and a signaling molecule, regulating transcriptional and physiological adaptations.^{[ 3} , ⁸ , ^{9 ]}

The initiation of nitrate signaling involves specialized transceptors: AtNRT1.1 in Arabidopsis and OsNRT1.1B in rice.^{[ 10} , ^{11 ]} Upon activation, these transceptors stimulate master transcriptional regulators—AtNLP7 in Arabidopsis and OsNLP3 in rice—by triggering their nuclear translocation to regulate the expression of nitrate‐responsive genes.^{[ 12} , ^{13 ]} However, the downstream cytoplasmic signaling mechanisms exhibit striking divergence between these two model plants.^{[ 14 ]} In Arabidopsis, nitrate perception by AtNRT1.1 elicits cytosolic Ca²⁺ fluxes that activate Ca²⁺‐sensor protein kinases (CPKs). These CPKs phosphorylate AtNLP7, stabilizing its nuclear localization and sustaining transcriptional activation of nitrate‐responsive genes.^{[ 15} , ^{16 ]} In contrast, rice utilizes a distinct ubiquitination‐mediated pathway: OsNRT1.1B perceives nitrate and subsequently recruits the E3 ligase OsNBIP1. This interaction triggers the degradation of the transcriptional repressor OsSPX4, thereby releasing OsNLP3 from cytoplasmic sequestration. As a result, OsNLP3 translocates to the nucleus, where it activates nitrate‐responsive genes.^{[ 13 ]}

Notably, AtNLP7‐mediated signaling cascade in Arabidopsis generates rapid transcriptional responses to N availability, characterized by distinct primary (minutes) and secondary (hours) phases that ultimately promote plant growth in response to nitrate.^{[ 6} , ¹⁷ , ^{18 ]} Despite this progress, the integration of these rapid signals with long‐term N utilization remains poorly understood. Therefore, establishing a unified nitrate signaling network that integrates rapid external nitrate sensing with adaptive responses for long‐term N utilization is critical for improving crop nitrogen use efficiency (NUE). Such a model would not only elucidate the mechanistic basis of primary nitrate response (PNR) but also provide actionable insights for sustainable agriculture.

Here, we address this critical gap by identifying a Ca²⁺‐dependent nitrate signaling pathway in rice that functions synergistically with the canonical ubiquitination cascade. We demonstrate that OsCNGC14 and OsCNGC16, two cyclic nucleotide‐gated channels, interact with OsNRT1.1B to transduce nitrate signal into intracellular Ca²⁺ fluxes. This Ca²⁺ signaling leads to the phosphorylation of OsNLP3 and amplifies its transcriptional activity. This dual regulatory mechanism enables rice to dynamically balance immediate nitrate sensing with sustained N utilization.

2. Results

2.1. A Calcium‐Dependent Pathway Complements the Ubiquitination Cascade in Rice Nitrate Signaling

Nitrate, an essential N source for plants, is well‐documented for its role in activating the PNR signaling pathway.^{[ 10} , ¹¹ , ¹⁵ , ¹⁷ , ¹⁹ , ^{20 ]} While numerous genes involved in PNR, including transporters, N assimilation enzymes, and transcription factors, have been implicated in regulating NUE,^{[ 11} , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ , ^{28 ]} the hierarchical organization of PNR‐controlled nitrate signaling in rice and its role in long‐term N utilization remain poorly understood. To address this, we performed time‐course RT‐qPCR analyses of nitrate‐responsive genes, revealing a biphasic induction pattern: an initial transcriptional peak at 1 h (PNR phase; Figure S1, Supporting Information) followed by a secondary peak at 4–5 h for OsNRT2.1, OsNIA, and OsNIR (except for OsGS1.1) (Figure S1A–C, Supporting Information). To dissect these two regulatory phases, we analyzed nitrate induction in the osnrt1.1b and osnbip1‐1 mutants. Strikingly, both induction peaks were abolished in osnrt1.1b (Figure S1, Supporting Information), whereas osnbip1‐1 mutant retained the secondary peak at levels comparable to wild‐type (Figure S1, Supporting Information). This indicates that osnbip1 specifically disrupts PNR without impairing the secondary nitrate response. Notably, this PNR‐independent secondary activation in rice diverges from the canonical NLP‐mediated cascade in Arabidopsis,^{[ 29 ]} suggesting the existence of a parallel regulatory pathway operating alongside the OsNRT1.1B‐OsSPX4‐OsNLP3 axis.

In Arabidopsis, it was reported that the dual‐function “transceptor” AtNRT1.1 mediates nitrate‐induced Ca²⁺ influx,^{[ 16} , ²⁰ , ^{30 ]} however, the role of calcium signaling in rice and its dependence on OsNRT1.1B remain unresolved. To investigate this, we used Zhonghua11 (ZH11) carrying the calcium indicator YC3.6 (ZH11^YC3.6) to cross with the osnrt1.1b mutant, producing the osnrt1.1b ^YC3.6. Application of 10 mm KNO₃ to root tips elicited robust Ca²⁺ spikes in ZH11^YC3.6 but not in osnrt1.1b ^YC3.6 (Figure 1A,B), demonstrating that OsNRT1.1B is essential for nitrate‐induced Ca²⁺ signaling in rice.

Figure 1.

OsNRT1.1B mediates nitrate‐triggered Ca²⁺ signaling to complement the ubiquitination cascade in nitrate signaling A). Time‐course analysis of KNO₃‐induced cytosolic Ca²⁺ ([Ca^{2 +}]_cyt) dynamics. Root tips of ZH11 and osnrt1.1b plants expressing the Ca²⁺ sensor YC3.6 were treated with 10 mm KNO₃ and 10 mm KCl served as a negative control. Images were acquired at 1‐s intervals. Black bars indicate the time‐points of when treatment was applied. Means ± SD. (n = 3 biological replicates). B). Quantification of nitrate‐triggered [Ca^{2 +}]_cyt elevation. Histogram indicates the average [Ca^{2 +}]_cyt levels in ZH11 and osnrt1.1b root tips treated by KNO₃ or KCl. Means ± SD. (n = 3 biological replicates). C). Calcium channel blockade suppresses expression of primary nitrate‐responsive genes. RT‐qPCR analysis of nitrate‐inducible genes (OsNRT2.1, OsNIA, and OsNIR) in 14‐day‐old ZH11 and OsSPX4‐OE1 roots treated with 5 mm KNO₃ for 30 min, with or without La³⁺ (Ca²⁺ channel inhibitor) pretreatment. Relative expression levels were normalized to the KCl‐treated ZH11 (control set as 1). Means ± SD. (n = 3 biological replicates; two‐tailed student's t‐test, ^** P < 0.01; ns, no significance).

Given the critical role of Ca²⁺ in PNR activation, we further analyzed nitrate‐responsive gene expression, RT‐qPCR revealed strong suppression of OsNIA, OsNIR, and OsNRT2.1 in OsSPX4‐OE1 and osnrt1.1b plants within 30 min of nitrate treatment (Figure 1C). Importantly, pretreatment with La³⁺, a plasma membrane Ca²⁺ channel blocker, significantly inhibited gene induction in ZH11 and OsSPX4‐OE1 plants but had minimal effect on osnrt1.1b mutant (Figure 1C). These findings indicate that both Ca²⁺ signaling and OsSPX4 degradation converge on OsNRT1.1B to orchestrate primary nitrate responses.

2.2. OsCNGC14/16 and OsNRT1.1B form a Nitrate‐Activated Calcium Channel

Cation channels mediating Ca²⁺ influx in plants belong to multiple protein families.^{[ 31 ]} Based on evidence that AtCNGC15 facilitates nitrate‐triggered Ca²⁺ signaling in Arabidopsis ^{[ 16 ]} and OsCNGCs enhance rice growth and yield,^{[ 32 ]} we systemically screened all 16 members of OsCNGC family to identify candidates mediating nitrate‐induced Ca²⁺ influx in rice. Using split‐ubiquitin yeast two‐hybrid (Y2H) assays, we identified interactions between OsNRT1.1B and OsCNGC1, 6, 10, 14, and 16 (Figure S2A, Supporting Information). RNA‐seq analysis of roots treated with 5 mm KNO₃ showed nitrate‐inducible expression of OsCNGC7, 8, 14, and 16 (Figure S2B, Supporting Information). Furthermore, hydroponic assays under varying N regimes identified OsCNGC3, 5, 8, 13, 14, 15, and 16 as being dynamically regulated in roots or shoots (Figure S2C, Supporting Information). Significantly, OsCNGC14 and 16 consistently interacted with OsNRT1.1B, exhibited nitrate‐responsive expression in both short‐ and long‐term treatments, and showed co‐expression patterns with OsNRT1.1B (Figure S2D–F, Supporting Information). These results collectively suggest OsCNGC14 and OsCNGC16 as prime candidates for mediating nitrate‐triggered Ca²⁺ influx in rice.

To determine whether OsCNGC14, OsCNGC16, and OsNRT1.1B form a functional complex akin to Arabidopsis AtNRT1.1‐AtCNGC15 “transceptor‐channel”,^{[ 16 ]} we further analyzed their spatial and physical interactions. RT‐qPCR analysis confirmed overlapping expression of OsCNGC14, OsCNGC16, and OsNRT1.1B in root tips (Figure 2A). They also co‐localized at the plasma membrane (Figure 2B), and Co‐IP and luciferase complementation imaging (LCI) assays further validated their interaction in planta (Figure 2C; Figure S3A, Supporting Information). Two‐Electrode Voltage Clamp (TEVC) recordings in Xenopus oocytes revealed that neither OsCNGC14 nor OsCNGC16 alone exhibited Ca²⁺ channel activity; however, when co‐expressed at a 1:1 ratio, they formed a functional heteromeric channel generating robust Ca²⁺ currents (Figure 2D,E). LCI and split‐ubiquitin Y2H assays confirmed heteromerization of OsCNGC14 and OsCNGC16 (Figure S3B,C, Supporting Information), which were co‐localized in oocyte membranes (Figure S3D, Supporting Information). Strikingly, co‐expression of OsNRT1.1B with OsCNGC14 and OsCNGC16 at a 1:1:1 ratio abolished these Ca²⁺ currents (Figure 2D,E), demonstrating that OsNRT1.1B inhibits the OsCNGC14/16 channel complex. Furthermore, ¹⁵N‐nitrate uptake assays confirmed that OsCNGC14 or OsCNGC16 neither facilitates nitrate transport nor influences the transport activity of OsNRT1.1B in oocytes (Figure S3E,F, Supporting Information), thereby underscoring their exclusive function in Ca²⁺ signaling.

Figure 2.

OsNRT1.1B‐OsCNGC14/16 complex functions as a nitrate‐responsive calcium channel A). Tissue‐specific expression of OsCNGC genes and OsNRT1.1B in rice root tips (≤5 mm). RT‐qPCR analysis of OsCNGC1/6/7/10/14/16 and OsNRT1.1B expression in ZH11 root tips. Relative transcript levels were normalized to the expression level of OsNRT1.1B (set as 1). Means ± SD. (n = 3 biological replicates). B). Subcellular co‐localization of OsCNGC14 and OsNRT1.1B, OsCNGC16 and OsNRT1.1B in rice protoplasts. Confocal images of 12‐day‐old rice protoplasts co‐expressing OsCNGC14‐mCherry and OsNRT1.1B‐GFP (top) or OsCNGC16‐mCherry and OsNRT1.1B‐GFP (bottom). Left, mCherry (561 nm excitation), middle, GFP (488 nm excitation), right, overlay (mCherry, GFP, and DIC) of the same sample. Scale bars = 10 µm. C). OsCNGC14 and OsCNGC16 physically interact with OsNRT1.1B in planta. Co‐immunoprecipitation (Co‐IP) assay in rice protoplasts co‐expressing OsCNGC14‐FLAG and OsNRT1.1B‐HA, OsCNGC16‐FLAG and OsNRT1.1B‐HA; FLAG and OsNRT1.1B‐HA was used as negative control. The ponceau S staining of Rubisco verifies equal protein loading in the input group. D). Nitrate‐regulated calcium channel activity of the OsNRT1.1B‐OsCNGC14/16 complex. Two‐electrode voltage‐clamp (TEVC) recordings from Xenopus laevis oocytes expressing OsNRT1.1B, OsCNGC14, OsCNGC16, OsCNGC14 + OsCNGC16, and OsCNGC14 + OsCNGC16 + OsNRT1.1B. Bath solutions containing 20 mm CaCl₂ + 10 mm KCl or 20 mm CaCl₂ + 10 mm KNO₃ (nitrate‐stimulated), by step‐wise model. Voltage steps: −140 to +20 mV (step 10 mV). Water‐injected was used as a negative control. E). Quantitative analysis of nitrate‐responsive calcium currents. Current amplitudes at −140 mV from multiple recordings in (D). Means ± SD. (n = 4 oocytes per group, one‐way ANOVA with Tukey's multiple comparisons test, P < 0.05).

Similar to the dual‐function nitrate sensor AtNRT1.1, OsNRT1.1B suppresses OsCNGC14/16 activity, forming a resting transceptor‐channel complex (Figure 2D,E). To examine whether nitrate perception influences this interaction, we treated oocytes co‐expressing OsNRT1.1B and OsCNGC14/16 with 10 mm nitrate. Nitrate treatment restored Ca²⁺ currents of OsNRT1.1B‐OsCNGC14/16 to levels approaching those observed with OsCNGC14/16 (Figure 2D,E). Consistent with this result, Co‐IP assays in rice protoplasts expressing OsCNGC14‐FLAG and OsNRT1.1B‐HA, OsCNGC16‐FLAG and OsNRT1.1B‐HA showed a strong basal interaction that was weakened upon nitrate treatment (Figure S3G, Supporting Information); LCl assays in tobacco leaves confirmed this result (Figure S3H,I, Supporting Information). Together, these findings define a nitrate‐triggered calcium channel formed by the heteromeric OsCNGC14/16 complex and OsNRT1.1B. Nitrate perception relieves OsNRT1.1B‐mediated inhibition, thereby activating the channel and initiating Ca²⁺ influx.

2.3. OsCNGC14 and OsCNGC16 are Essential for Nitrate‐Induced Calcium Signaling and Nitrogen Utilization in Rice

To investigate whether OsCNGC14 and OsCNGC16 are involved in nitrate‐induced calcium signaling, we generated loss‐of‐function mutants, oscngc14 and oscngc16, using the CRISPR/Cas9 system (Figure S4, Supporting Information). These mutants were subsequently crossed with the ZH11^YC3.6 to produce the oscngc14 ^YC3.6 and oscngc16 ^YC3.6 lines. Nitrate‐triggered Ca²⁺ influx was monitored in root tips by treating with 10 mm KNO₃. A robust calcium spike was observed in ZH11^YC3.6 roots, but not in oscngc14 ^YC3.6, oscngc16 ^YC3.6, and osnrt1.1b ^YC3.6 lines (Figure 3A–C). Notably, NaCl‐induced Ca²⁺ influx remained unaffected in all lines (Figure 3B,C), demonstrating that OsCNGC14 and OsCNGC16 are essential for generating nitrate‐triggered elevations in intracellular calcium concentration ([Ca^{2 +}]_i) in root tip cells.

Figure 3.

The oscngc14 and oscngc16 display abolished Ca²⁺‐dependent PNR and reduced long‐term expression of nitrogen assimilation genes. A). Images of nitrate‐triggered cytosolic Ca²⁺ ([Ca^{2 +}]_cyt) in root tips. Time‐lapse pseudo‐color ratio imaging of [Ca^{2 +}]_cyt in ZH11, oscngc14, and oscngc16 root tips were treated with 10 mm KNO₃. Time‐point of 0 s indicates treatment initiation. Red circles denote regions of root tips used for ratiometric measurements. The [Ca^{2 +}]_cyt responses were visualized using a color gradient from low (blue) to high (red). Scale bar = 50 µm. B). Time‐course analysis of nitrate‐induced [Ca^{2 +}]_cyt fluxes. ZH11, osnrt1.1b, oscngc14, and oscngc16 root tips expressing the Ca²⁺ sensor YC3.6 were treated with 10 mm KCl (left, negative control, n = 3 biological replicates), 10 mm KNO₃ (middle, n = 4 biological replicates), and 100 mm NaCl (right, osmotic control, n = 3 biological replicates). Images were acquired at 1 s intervals. Black bar indicates the time‐points of treatment applied. Means ± SD. C). Quantitative analysis of [Ca^{2 +}]_cyt responses. Histogram indicates the average [Ca^{2 +}]_cyt of ZH11, osnrt1.1b, oscngc14, and oscngc16 root tips treated by KCl, KNO₃, and NaCl in (B). Means ± SD. (n ≥ 3 biological replicates, two‐tailed Student's t‐test ** P < 0.01; ns, no significance). D). The mutants of OsCNGC14 and OsCNGC16 blockade suppresses expression of primary nitrate‐responsive genes. RT‐qPCR analysis of primary nitrate‐inducible genes (OsNRT2.1, OsNIA, and OsNIR) in 14‐day‐old ZH11, osnrt1.1b, oscngc14, and oscngc16 roots treated with 10 mm KNO₃ or KCl for 30 min, with or without LaCl₃ pretreatment. Means ± SD. (n = 3 biological replicates; two‐tailed student's t‐test, ^* P < 0.05, ** P < 0.01; ns, no significance). E). RT‐qPCR analysis of N‐related genes (OsNRT2.1, OsNIA, OsNIR, OsGS1.1, OsNLP1, OsNLP3, and OsNLP4) in 60‐day‐old of ZH11, osnrt1.1b, oscngc14, and oscngc16 roots which were grown in greenhouse. Means ± SD. (n = 3 biological replicates sample; one‐way ANOVA with Tukey's multiple comparisons test, P < 0.05).

Next, we assessed the role of OsCNGC14 and OsCNGC16 in Ca^{2 +}‐dependent nitrate‐responsive gene expression using RT‐qPCR analysis. In roots, the expression of key target genes, OsNRT2.1, OsNIA, and OsNIR, was reduced in osnrt1.1b, oscngc14, and oscngc16 mutants (Figure 3D). These reductions mirrored the effect observed in ZH11 roots pretreated with the Ca²⁺ channel blocker LaCl₃. Under infertile‐soil culture in a greenhouse, N assimilation genes (OsNIA, OsNIR, OsGS1.1) and the transcription factor OsNLP3 were significantly downregulated in osnrt1.1b, oscngc14, and oscngc16 mutants, while other nitrate‐responsive transcription factors remained unchanged (Figure 3E). Combined with previous reports of growth inhibition in osnlp3 mutant,^{[ 27 ]} these findings suggest that calcium‐dependent PNR may regulate long‐term expression of N‐related genes in rice via OsNLP3 expression.

Field trials conducted under low‐ and normal‐N conditions demonstrated severe growth defects in oscngc14 and oscngc16 mutants, including reduced plant height, panicle length, tiller number, and ultimately lower grain yield compared to ZH11 under both N regimes, which was similar to the osnrt1.1b mutant (Figure S5A–C, Supporting Information). Hydroponic assays further confirmed impaired shoot development, root architecture establishment, and reduced biomass accumulation in osnrt1.1b, oscngc14, and oscngc16 mutants under insufficient and sufficient N supplies (Figure S5D,E, Supporting Information). Moreover, ¹⁵N‐nitrate or ¹⁵N‐ammonium feeding experiments showed reduced nitrate uptake and root‐to‐shoot transport in these mutants, while ammonium utilization was less affected. This reduction ultimately resulted in a decrease of total N content in both roots and shoots (Figure S5F,G, Supporting Information). Coupled with the altered expression of nitrogen‐related genes (Figure 3E), this disturbance of N homeostasis led to reduced grain yield and NUE in osnrt1.1b, oscngc14, and oscngc16 mutants. Notably, OsSPX4‐OE1 and osnbip1‐1 plants exhibited PNR repression similar to that of osnrt1.1b, oscngc14, and oscngc16 mutants (Figures 1, 3; Figure S1, Supporting Information). However, unlike these mutants, OsSPX4‐OE and osnbip1 plants showed only slight reductions in shoot growth, root development, biomass, N uptake, and N content in hydroponic or ¹⁵N feeding experiments (Figure S6A–D, Supporting Information). Furthermore, OsSPX4‐OE1 plants showed no significant growth inhibition in the field, particularly in tiller number (Figure S6E, Supporting Information). These results suggest that nitrate‐induced Ca²⁺ signaling regulates both PNR and long‐term nitrate responses through a pathway distinct from the OsSPX4 degradation cascade.

2.4. Phosphorylation of OsNLP3 at Ser193 Links Nitrate‐Induced Calcium Signaling and Transcriptional Activation

OsNLP3, a key transcription factor, is released from OsSPX4 repression upon nitrate exposure.^{[ 13 ]} However, it remains unclear whether OsNLP3 undergoes nitrate‐induced activation via phosphorylation—similar to AtNLP7/6 in Arabidopsis.^{[ 15 ]} To address this, we utilized OsNLP3‐FLAG transgenic plants to test OsNLP3 protein modification in phos‐tag assays. Interestingly, it revealed that nitrate triggers the phosphorylation of OsNLP3, as the effect was abolished by λ phosphatase treatment (Figure S7A, Supporting Information). Furthermore, pretreatment with LaCl₃ dramatically reduced this phosphorylation (Figure S7B, Supporting Information). Sequence alignment identified a conserved phosphorylation residue at serine 193 (Ser193)^{[ 15 ]} within the GAF domain across NLPs in both Arabidopsis and rice (Figure S7C, Supporting Information). This residue was also predicted by prediction tools (NetPhos‐3.1). Mutational analysis further confirmed the functionality of this residue: a phosphoablative variant (OsNLP3^S193A) exhibited attenuated phosphorylation, whereas a phosphomimetic variant (OsNLP3^S193D) displayed constitutive phosphorylation (Figure S7D, Supporting Information). Together, these findings establish Ser193 as the primary residue of nitrate‐induced phosphorylation.

Next, we investigated how phosphorylation influences OsNLP3 activity by examining its subcellular localization in rice protoplasts. Following nitrate stimulation, both eGFP‐OsNLP3 and eGFP‐OsNLP3^S193D rapidly translocated to the nucleus within 45 min. In contrast, the eGFP‐OsNLP3^S193A showed delayed nuclear accumulation, requiring 90 min (Figure S8A, Supporting Information). This observation was confirmed by protein degradation assays and nuclear‐cytoplasmic fractionation (Figure S8B,C, Supporting Information), suggesting that phosphorylation at Ser193 accelerates the nuclear translocation of OsNLP3. Luciferase assays demonstrated that OsNLP3^S193D enhanced transcriptional activation of key target genes, OsNRT2.1, OsNIA, and OsGS1.1, while OsNLP3^S193A increased OsNIR expression (Figure S9A, Supporting Information). Importantly, both the phosphoablative and phosphomimetic variants of OsNLP3 acted as positive regulators of target genes. Moreover, when complemented in osnlp3 protoplasts, all three forms (OsNLP3‐FLAG, OsNLP3^S193A‐FLAG, or OsNLP3^S193D‐FLAG) showed comparable protein abundance (Figure S10, Supporting Information) and similarly upregulated target genes (Figure S9B, Supporting Information). These findings indicate that although phosphorylation of Ser193 enhances OsNLP3 nuclear translocation or transcriptional activity, it is not essential for these processes. This provides regulatory flexibility critically for rice adaptation to fluctuating nitrate availability, unlike the mandatory requirement seen for AtNLP7 activation.

Consistent with disrupted Ca²⁺ signaling in osnrt1.1b, oscngc14, and oscngc16 mutants (Figure 3B), we also observed abolished OsNLP3 phosphorylation and delayed nuclear translocation in these mutants (Figure 4A,B), directly linking Ca²⁺ signaling to OsNLP3 activation. While OsNLP3 transcript levels increased 2 h after N starvation following nitrate resupply,^{[ 27 ]} this induction was absent in osnrt1.1b, oscngc14, and oscngc16 mutants, with only a delayed response observed in the osnbip1 mutant (Figure 4C). Transactivation assays revealed OsNLP3^S193D strongly activated its own promoter, whereas the OsNLP3^S193A variant showed minimal activity (Figure 4D). This result indicates that Ca²⁺ signaling is essential for activating the transcription of OsNLP3 in response to nitrate. Accordingly, RT‐qPCR verified that nitrate‐responsive genes were not activated in the roots of osnrt1.1b, oscngc14, and oscngc16 mutants within 4–6 h of treatment (Figure S11, Supporting Information). In summary, our results show that nitrate‐triggered calcium influx starts a signaling cascade that leads to the phosphorylation of OsNLP3 at Ser193, allowing its self‐activation and promoting the expression of nitrate‐responsive genes. (Figure 4E).

Figure 4.

OsNRT1.1B‐OsCNGC14/16‐mediated phosphorylation triggers accelerated nuclear translocation and enhances transcriptional activation of OsNLP3. A) Nitrate‐induced OsNLP3 phosphorylation depends on OsNRT1.1B, OsCNGC14, and OsCNGC16. Phos‐tag and SDS‐PAGE analysis of OsNLP3‐FLAG phosphorylation in protoplasts from ZH11, osnrt1.1b, oscngc14, and oscngc16 plants treated with 10 mm KNO₃ or KCl for 30 min. Phosphorylation intensity (normalized to KCl‐treated ZH11, set as 1) was quantified from 8% phos‐tag gels (top). Total protein levels were verified by 10% SDS‐PAGE (middle). Actin serves as loading control (bottom). B) OsNRT1.1B‐OsCNGC14/16 accelerates nuclear translocation of OsNLP3 under nitrate treatment. Confocal images of protoplasts co‐expressing eGFP‐OsNLP3 and HY5‐mCherry from 12‐day‐old ZH11, osnrt1.1b, oscngc14, and oscngc16 plants. The protoplasts were treated with 5 mm KNO₃ or KCl for 45 min. GFP fluorescence (488 nm excitation) (top). Overlay (GFP, mCherry and DIC) of the same sample (bottom). Scale bars = 5 µm. C) Calcium signaling sustains OsNLP3 transcriptional activation. Time‐course RT‐qPCR of OsNLP3 expression in ZH11, osnbip1‐1, osnrt1.1b, oscngc14, and oscngc16 roots treated with 5 mm KNO₃ or KCl for 0–8 h. Data are normalized to KCl‐treated controls (set as 1). Means ± SD. (n = 3 biological replicates). D). Phosphorylation of OsNLP3 at Ser193 confers OsNLP3 transcriptional activation. Transactivation assays of OsNLP3‐FLAG, OsNLP3^S193A‐FLAG or OsNLP3^S193D‐FLAG with the promoters of OsNLP3 were conducted. The dual‐luciferase reporter plasmids driven by OsNLP3 promoter were transiently expressed in osnlp3 protoplasts together with empty vector (control) or OsNLP3 effectors (OsNLP3‐FLAG, OsNLP3^S193A‐FLAG, or OsNLP3^S193D‐FLAG), respectively. Luciferase activity was measured at 30–180 min after treatment with 5 mM KNO₃. Data are normalized to OsNLP3‐FLAG control (set as 1). Means ± SD. (n = 3 biological replicates). E). Nitrate signaling network integrates with calcium signaling and canonical ubiquitination cascade in rice. (1) OsNRT1.1B‐OsCNGC14/16‐Ca²⁺ pathway: nitrate‐triggered calcium influx phosphorylates OsNLP3 (Ser193), enabling its rapid nuclear translocation and higher transcriptional autoactivation. (2) OsNRT1.1B‐OsNBIP1‐OsSPX4 pathway: ubiquitination‐mediated OsSPX4 degradation releases OsNLP3 for sustained N assimilation.

3. Discussion

Plants have evolved distinct nitrate signaling pathways to adapt to fluctuating N availability, as illustrated by the OsNRT1.1B‐OsNBIP1‐OsSPX4‐OsNLP3 pathway in rice and the AtNRT1.1‐Ca²⁺‐AtCPKs‐AtNLP7 pathway in Arabidopsis.^{[ 13} , ¹⁵ , ^{16 ]} Comparative analyses of these systems provide critical insights into the evolutionary divergence of nitrate‐sensing mechanisms. In this study, we identified the OsNRT1.1B‐OsCNGC14/16 complex as a dual‐function transceptor‐channel that senses elevated nitrate levels and triggers calcium influx to initiate downstream signaling (Figure 4E).

To dissect the roles of these pathways, we compared the growth phenotypes and N homeostasis of related mutants and overexpression plants in two signaling pathways under low and normal N conditions. Although the two pathways regulate different developmental processes and aspects of N homeostasis, the adverse effects of disrupted nitrate signaling on growth and N regulation become less severe under higher N conditions (Figures S5 and S6, Supporting Information). These findings suggest that elevated nutrient levels attenuate the influence of nitrate signaling on growth and development, especially in root architecture establishment. This mechanism explains how the yield penalty is alleviated in oscngc14 and oscngc16 mutants under normal N supply (Figure S5A,B, Supporting Information). Our results imply that enhancing nitrate signaling could significantly improve NUE in N‐limited soils.

Notably, rice appears to employ more complex layers of regulation than Arabidopsis. While calcium signaling is essential for sustained nitrate responses, OsNLP3 retains partial Ca²⁺‐independent activity, suggesting that rice integrates both canonical and lineage‐specific mechanisms in nitrate signaling. Rather than substituting the canonical ubiquitination pathways in rice, Ca²⁺ signaling collaborates with the OsSPX4‐mediated repression system, thereby facilitating the precise regulation of N metabolism under fluctuating environmental conditions. This dual‐layer regulation, combining rapid Ca²⁺‐dependent phosphorylation with the adjustable interactions between OsSPX4 and OsNLP3, may underpin rice's exceptional adaptability to fluctuating nutrient availability.

Beyond their role in N signaling, OsCNGCs affect plant growth and responses to various abiotic stresses, including ABA signaling, reactive oxygen species (ROS), and temperature fluctuations.^{[ 33} , ³⁴ , ³⁵ , ^{36 ]} Intriguingly, OsCNGC14 and OsCNGC16 participate in both ABA and temperature signaling and nitrate‐triggered Ca^{2 +} influx.^{[ 35} , ^{36 ]} Although the hypersensitivity of the oscngc14/16 double mutant to cold suggests functional redundancy between the two members in temperature signaling,^{[ 35 ]} the molecular basis for their interaction remains unclear. Our evidence indicates that OsCNGC14 and OsCNGC16 form a heteromeric complex specifically responsible for nitrate‐triggered calcium influx (Figure 2D,E, and 3A‐C). Mechanistically, these channels appear to operate via different activation modes: 1) In temperature signaling, they may function similarly to AtCNGC5 and AtCNGC6 in the cold response,^{[ 37 ]} potentially exhibiting functional redundancy; 2) In nitrate signaling, they resemble AtCNGC15,^{[ 16 ]} working cooperatively with AtNRT1.1 to form a transceptor‐channel complex for nitrate‐triggered calcium signal encoding. Therefore, our discovery of the OsNRT1.1B‐OsCNGC14/16 interaction establishes a direct mechanistic link between nutrient availability and stress resistance.

Among the 16 OsCNGCs in rice, only a subset, including OsCNGC14 and OsCNGC16, has defined roles in signaling or ion transport.^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ^{38 ]} Our investigation reveals that multiple OsCNGCs associate with OsNRT1.1B, although their functional relevance remains to be characterized. Given the established role of OsNRT1.1B in nitrate/phosphate signaling and ammonium utilization, these associated OsCNGCs may mediate additional processes such as auxin transport or root development, as seen in Arabidopsis.^{[ 39 ]} Future studies should explore whether OsCNGCs act as versatile signaling hubs that coordinate nutrient sensing with developmental processes.

In conclusion, our data delineate two interconnected pathways governing nitrate responses in rice: 1) The OsNRT1.1B‐OsNBIP1‐OsSPX4‐OsNLP3 axis fine‐tunes N metabolism in response to short‐term nitrate fluctuations (minutes to 2–3 h), ensuring a baseline toward N supply; 2) The OsNRT1.1B‐OsCNGC14/16‐Ca²⁺‐OsNLP3 pathway enables rapid detection (seconds to minutes) of external nitrate surges and sustains long‐term N homeostasis (3–4 h to weeks) through phosphorylation‐mediated mechanisms. These pathways allow rice to dynamically balance immediate nutrient acquisition with long‐term metabolic adjustments. Our work not only advances mechanistic understanding of nitrate signaling but also identifies conserved nodes at the intersection of nutrient sensing and stress response as promising targets for improving crop resilience.

4. Experimental Section

Plant Materials and Growth Conditions

The wild‐type rice Zhonghua 11 (O. sativa L. japonica, ZH11) was used in this study. The mutants of osnrt1.1b, osnbip1‐1, osnbip1‐2, and osnlp3, as well as the overexpression lines of OsSPX4‐OE1, OsSPX4‐OE2, and OsNLP3‐FLAG‐OE were previously generated in the laboratory. The oscngc14 and oscngc16 mutants were generated in the ZH11 background using CRISPR/Cas9‐mediated gene editing. For cytosolic imaging assays, osnrt1.1b, oscngc14, and oscngc16 mutants were crossed with YC3.6‐expressing plants (ZH11^YC3.6). For hydroponic culture (1–3 weeks; used for nitrate induction experiments, gene expression analysis, and rice protoplast preparation), rice seedlings were grown in a growth chamber with a 16‐h light/8‐h dark photoperiod, light intensity of ≈200 µmol m⁻² s⁻¹, temperature of 28/25 °C (day/night), and humidity of ≈70%. The basic nutrient solution contained the following macronutrients (in mm): (NH₄)₂SO₄ (0.5), MgSO₄·7H₂O (0.54), CaCl₂·2H₂O (0.36), K₂SO₄ (0.1), KH₂PO₄ (0.18), and Na₂SiO₃·9H₂O (1.6). The micronutrient composition (in µm) was: MnCl₂·4H₂O (9.14), H₃BO₃ (46.2), (NH₄)₆Mo₇O₂₄·4H₂O (0.08), ZnSO₄·7H₂O (0.76), CuSO₄·5H₂O (0.32), and Fe(II)‐EDTA (40), adjusted to pH 5.7. The nutrient solution was replaced daily. Different concentrations of KNO₃ were added to the basic solution depending on experimental treatments. For long‐term greenhouse cultivation (2–3 months; used for detecting long‐term root N‐responsive gene expression), rice plants were grown in a greenhouse under low N condition: 5 g of N (mixture of 60% nitrate, 40% ammonium) per 1 m² with a 12‐h light/12‐h dark photoperiod, natural light intensity, temperature of 30/24 °C (day/night), and humidity of ≈40–60%.

Vector Construction and Generation of Transgenic Rice

To generate oscngc14 and oscngc16 mutants, the 20‐nt small guiding RNA was cloned into CRISPR/Cas‐BGK03 (Biogle).^{[ 40 ]} Then the vectors were introduced into the Agrobacterium strain EHA105. The wild‐type ZH11 was used as the recipient for Agrobacterium‐mediated transformation, as previously described, to generate the transgenic rice.^{[ 41 ]} All constructs were verified by sequencing. T3 homozygous plants were used for subsequent experiments. For split‐ubiquitin analysis, the full‐length coding sequence (CDS) of OsNRT1.1B and OsCNGCs were cloned to generate either the pPR3‐N (OsNRT1.1B, OsCNGC16) or pMET (OsCNGCs). For studying protein interactions in Nicotiana benthamiana using OsCNGC14‐nLUC, OsCNGC16‐nLUC, cLUC‐OsNRT1.1B, and cLUC‐OsCNGC16 constructs, CDS of OsNRT1.1B, OsCNGC14, and OsCNGC16 were amplified and cloned into pCAMBIA1300‐nLUC and pCAMBIA1300‐(ATG)‐cLUC vectors. To observe co‐immunoprecipitation (Co‐IP) in protoplasts and subcellular localization in protoplasts and Xenopus oocytes using OsNRT1.1B‐GFP/HA, OsCNGC14‐mCherry/GFP/FLAG, OsCNGC16‐mCherry/FLAG, AtHY5‐mCherry and eGFP‐OsNLP3, CDS of OsNRT1.1B, OsCNGC14, OsCNGC16 from ZH11 and AtHY5 from Arabidopsis thaliana ecotype Col‐0 were amplified and cloned into pGEMHE‐eGFP, pGEMHE‐mCherry, pSAT1‐35S‐eGFP/mCherry, pCAMBIA2300‐35S‐HA, pCAMBIA2300‐35S‐FLAG, and pCAMBIA2300‐35S‐eGFP‐N vectors. Site‐directed mutagenesis was used to generate pCAMBIA2300‐35S‐eGFP‐OsNLP3^S193A and pCAMBIA2300‐35S‐eGFP‐OsNLP3^S193D constructs. For electrophysiological assay in Xenopus oocytes using OsNRT1.1B, OsCNGC14, and OsCNGC16, CDS of OsNRT1.1B, OsCNGC14, and OsCNGC16 from ZH11 were amplified and cloned into pGEMHE vectors. To examine OsNLP3 phosphorylation and the effects of phosphorylated/non‐phosphorylated OsNLP3 on N‐responsive gene activation, CDS sequences of OsNLP3 and promoter sequences (2 kb upstream of ATG) of pOsNRT2.1, pOsNIA1, pOsNIR1, pOsGS1.1, and pOsNLP3 were cloned into pCAMBIA2300‐35S‐FLAG and pGreenII0800‐ubiq‐LUC vectors. Site‐directed mutagenesis was used to generate pCAMBIA2300‐35S‐OsNLP3^S193A‐FLAG and pCAMBIA2300‐35S‐OsNLP3^S193D‐FLAG constructs.

Nitrate Induction Assay of Nitrate‐Responsive Genes

Rice seedlings were grown in a basic nutrient solution containing 0.5 mm (NH₄)₂SO₄ for 2 weeks. Prior to the nitrate Induction assay, seedlings were pretreated with 0.5 mm (NH₄)₂SO₄ in basic solution for 48 h under continuous light (including the 2‐week growth period), and were pretreated with or without 2 mm LaCl₃ for 10 min, then transferred to a fresh solution supplemented with 5 mm KNO₃ or KCl. Roots were collected at specific time‐points for gene expression analysis (n = 3 replicates, 6–8 seedlings per replicate).

Yeast Two‐Hybrid Assays

pPR3‐N (OsNRT1.1B, OsCNGC16) and pMET (OsCNGCs) vectors were introduced into yeast strain NMY51 using the lithium acetate method. The NMY51 transformants were selected on SD medium lacking Leu and Trp for 3 days at 28 °C. Then the haploid cells of NMY51 were selected on SD medium containing 5 mm 3‐AT, lacking Leu, Trp, His, and Ade for 5 days at 28 °C. The combinations in this experiment were N:APP/pMET:Fe65 as positive control; pPR3‐N:OsNRT1.1B/pMET‐EV and pPR3‐N:OsCNGC16/pMET‐EV as negative controls.

RNA Isolation and qPCR Analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse‐transcribed with ReverTra Ace qPCR RT Master Mix (Toyobo). qPCR was performed using SYBR Green Master Mix (Toyobo) on a Chromo4 system (Bio‐Rad). Data were analyzed with Excel (Microsoft). Rice Actin1 served as the internal reference. Primers are listed in Table S1 (Supporting Information).

RNA‐Seq Analysis

Approximately 100 ZH11 seedlings per treatment were hydroponically grown as described above. For nitrate induction, seedlings pretreated with 1 mm ammonium were exposed to 5 mm KNO₃ or KCl. Samples were collected at 0, 1.75, 2.75, and 24 h (n = 3 replicates, 15 seedlings per replicate). For long‐term nitrate treatments, seedlings were cultured in modified Kimura B solution (0, 0.2, 2, and 10 mm KNO₃) with adjusted potassium concentrations. Roots and shoots of 14‐day‐old seedlings were collected for RNA‐seq (n = 3 replicates, 15 seedlings per replicate). BGI‐Wuhan performed library construction and sequencing.

Subcellular Localization Assay

All subcellular localization experiments used nitrate‐free rice protoplasts. Protoplasts were isolated from 12‐day‐old seedlings and transformed as previously described.^{[ 42 ]} To investigate the co‐localization of OsNRT1.1B with OCNGC14 or OsCNGC16. Nitrate‐free rice protoplasts were co‐transfected with pSAT1‐35S‐OsNRT1.1B‐mCherry and either pSAT1‐35S‐OsCNGC14‐GFP or 35S‐OsCNGC16‐GFP. To study the effect of osnrt1.1b, oscngc14, and oscngc16 on OsNLP3 localization under nitrate induction, nitrate‐free protoplasts were co‐transfected with pCAMBIA2300‐35S‐eGFP‐OsNLP3 and pSAT1‐35S‐AtHY5‐mCherry (1:0.8 ratio). To test the effect of Ser193 on OsNLP3 localization under nitrate induction, nitrate‐free protoplasts were co‐transfected with pCAMBIA2300‐35S‐eGFP‐OsNLP3/35S‐eGFP‐OsNLP3^S193A /35S‐eGFP‐OsNLP3^S193D and pSAT1‐35S‐AtHY5‐mCherry (1:0.8 ratio). Incubate the rice protoplasts transfected with the plasmid in W5 solution for 12 h. Protoplasts were centrifuged at 200 × g for 3 min, treated with W5 solution containing 5 mm KNO₃ for 0, 45, and 90 min, and observed under a confocal microscope (LSM 980, Carl Zeiss).

Luciferase Complementation Imaging (LCI) Assay

LCI assays were conducted to study interactions between OsCNGC14‐nLUC/cLUC‐OsNRT1.1B, OsCNGC16‐nLUC/cLUC‐OsNRT1.1B, and OsCNGC14‐nLUC/cLUC‐OsCNGC16. Agrobacterium strain GV3101 carrying constructs was cultured in LB medium at 28 °C for 16 h. Cells were pelleted, resuspended in infiltration buffer (10 mm MES (pH 5.6), 10 mm MgCl₂, 0.2 mM acetosyringone; OD₆₀₀ = 1.0), and incubated at room temperature for 3 h. Equal volumes of bacterial suspensions were co‐infiltrated into N. benthamiana leaves. Plants were kept at 23 °C for 48 h (16‐h light/8‐h dark). Luciferin (1 mm) and 0.01% Triton X‐100 (v/v) were sprayed onto leaves, and luminescence was captured using a low‐light CCD camera (NightOWL II LB983).

For LCI assays, the effect of nitrate on the interactions between OsNRT1.1B and OsCNGC14, OsNRT1.1B and OsCNGC16 was examined. The infiltrated leaves were first treated with 10 mm KNO₃ or KCl for 30 min, and then treated as described above.

Co‐Immunoprecipitation (Co‐IP) Assay

Nitrate‐free protoplasts co‐expressed with OsCNGC14‐FLAG + OsNRT1.1B‐HA and OsCNGC16‐FLAG + OsNRT1.1B‐HA were incubated in W5 solution for 12 h, centrifuged at 200 × g, and treated with 10 mm KNO₃ or KCl for 30 min. Total proteins were extracted from lysed protoplasts using 200 µL extraction buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 1% (v/v) Triton X‐100, 10% (v/v) glycerol, 1× EDTA‐free protease inhibitor cocktail). A 20 µL aliquot was mixed with 5 µL 6× SDS loading buffer as input. The remaining lysate was diluted to 1 mL with extraction buffer (without Triton X‐100), incubated with 20 µL anti‐FLAG magnetic beads (Lablead PFM050) at 4 °C for 2 h. Beads were washed three times with buffer (50 mm HEPES (pH 7.5), 150 mm NaCl), eluted with 2× SDS loading buffer at 55 °C for 10 min, and analyzed by SDS‐PAGE and immunoblotting using HRP‐conjugated anti‐FLAG or anti‐HA antibodies.

Two‐Electrode Voltage Clamp (TEVC) and Xenopus Oocyte ¹⁵N Uptake Assay

Full‐length CDS of OsCNGC14, OsCNGC16, and OsNRT1.1B were cloned into pGEMHE. Capped RNAs (cRNAs) were synthesized using the mMACHINE T7 Kit (Thermo) and stored at −80 °C. Stage V‐VI Xenopus oocytes were defolliculated with collagenase A (1.5 mg mL⁻¹ in Ca²⁺‐free ND96) at 26 °C, 40 rpm for 3 h, washed, and maintained in ND96 at 18 °C. Oocytes were injected with cRNA mixtures (e.g., 32 nL of 500 ng µL⁻¹ for OsCNGC14 and OsCNGC16; 500 ng µL⁻¹ for OsNRT1.1B). For TEVC, currents were recorded 2 days post‐injection using a GeneClamp 200B amplifier (Axon Instruments). Bath solutions contained 20 mm CaCl₂ + 10 mm KCl and 20 mm CaCl₂ + 10 mm KNO₃ adjusted osmotic to 220 mmol kg⁻¹ with mannitol. Voltage steps (−140 mV to +20 mV, 10 mV increments) were applied for 3 s. For ¹⁵ N uptake, oocytes were incubated in ¹⁵ N‐KNO₃‐containing solutions (0.2 or 10 mm ¹⁵ N‐KNO₃) for 6 h, then washed 5 times, dried at 65 °C for 2 days, and analyzed using an isotope mass spectrometer (DELTA V Advantage, Thermo Fisher).

In Vivo Degradation Assay

Protoplasts expressing target proteins pre‐cultured in 10 mm KCl or KNO₃ were treated with 200 µm cycloheximide (CHX) for 0, 45, 90, and 135 min. Pelleted protoplasts were extracted with lysis buffer (25 mm Tris‐HCl (pH 7.5), 1 mm EDTA, 150 mm NaCl, 1% (v/v) Triton X‐100, 5% (v/v) glycerol, and 1x protease and phosphatase inhibitor cocktail). Protein samples were analyzed by SDS‐PAGE and immunoblotting using anti‐FLAG or anti‐Actin antibodies. The X‐ray film was scanned and analyzed for statistics using ImageJ software.

Cytosol and Nuclear Fractions from Rice Protoplasts

The cytosol and nuclear fractions from plant tissue were separated according to the method described previously.^{[ 43 ]} After treatment with 10 mm KNO₃, pelleted protoplasts expressing target proteins were gently resuspended in 200 µL ice‐cold lysis buffer (20 mm Tris‐HCl (pH 7.4), 20 mm KCl, 2.0 mm EDTA, 25% (v/v) glycerol, 2.5 mm MgCl₂, 250 mm sucrose, 1x protease and phosphatase inhibitor cocktail) and incubated on ice for 10 min. The homogenate was filtered through a 100 and 40 µm nylon mesh sequentially. Lysates were clarified by centrifugation at 1500 × g for 20 min at 4 °C, and the supernatant was transferred to a new tube, the nuclei pellet was kept in the tube. The supernatant was further clarified by centrifugation at 20000 × g for 20 min at 4 °C and was designated as the cytosol fraction. The nuclei pellet fraction was gently resuspended in 1 mL NRBT buffer (20 mm Tris‐HCl (pH 7.4), 2.5 mm MgCl₂, 25% (v/v) glycerol, 0.2% (v/v) NP40, 0.1% (v/v) Triton X‐100), and centrifuged at 1500 × g for 10 min at 4 °C. The nuclei fraction was washed twice using the NRBT buffer. Finally, the nuclear pellet was resuspended in 100 µL total protein extraction buffer (50 mm Tris‐HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1% (v/v) Triton X‐100, 1x protease and phosphatase inhibitor cocktail), and then eluted by boiling in reducing SDS loading buffer. The cytosolic and nuclear fractions were resolved by SDS‐PAGE and immunoblotted with anti‐FLAG antibody, and anti‐UGPase and anti‐H3 antibodies served as compartment‐specific markers for cytosol and nucleus, respectively. The X‐ray film was scanned and analyzed for statistics using ImageJ software.

Cytosolic [Ca²⁺] Imaging Using Yellow Cameleon

Seeds of ZH11 ^YC3.6, osnrt1.1b ^YC3.6, oscngc14 ^YC3.6, and oscngc16 ^YC3.6 were sterilized and grown on nitrate‐free Kimura medium (0.5 mm (NH₄)₂SO₄, 0.6% agar, pH 5.7) for 12–16 days. Ratiometric Ca²⁺ imaging was performed using a Zeiss LSM 710 confocal microscope (excitation: 445 nm; emission: 484–505 nm for CFP, 520–528 nm for FRET).

Field Cultivation of Rice

To investigate the effects of oscngc14 and oscngc16 mutants on N utilization in the field, three large‐scale field trials were conducted from 2022 to 2024 at two experimental stations: the Institute of Genetics and Developmental Biology (IGDB, Beijing) and the IGDB South China Experimental Station (Lingshui, Hainan).

2022 Hainan Trial (2021 December‐April): N source: a mixture of 60% nitrate, 40% ammonium. Low N treatment: 0.5 kg N per 100 m²; normal N treatment: 1 kg N per 100 m². Fertilizers: KNO₃ (nitrate source) and (NH₄)₂SO₄ (ammonium source). Phosphorus fertilizer: P₂O₅ (0.5 kg P per 100 m²) applied before transplanting. Fertilizer uniformity: N fertilizers were evenly distributed to minimize variability.

2022 Beijing Trial (May‐October): N source: same as above. Low and normal N treatments are identical to Hainan cultivation.

2024 Hainan Trial (2023 December–April): Conditions are identical to the 2022 Hainan and 2022 Beijing cultivation. For large‐scale field tests, the plot size for yield tests was 1.44 m², with each plot containing 36 effective plants. The calculation formula of NUE is as follows: Actual yield per plot (g) / [area of plot (m²) x N fertilizer applied (kg)].

To investigate OsSPX4‐overexpressing plants grown in rice under field conditions, on field trial was conducted in 2024 at the Institute of Genetics and Developmental Biology (IGDB, Beijing). Low N condition was identical to 2022 Beijing cultivation.

In all field trials, planting spacing: 20 cm. Irrigation and design: Continuous flooding maintained throughout growth; completely randomized block design for each plot. Planting density: 8 rows × 8 plants per plot. Border rows were removed before final analysis to mitigate edge effects.

Labeling with ¹⁵N‐Nitrate and ¹⁵N‐Ammonium for Determining ¹⁵N Accumulation Assay in Plants

Rice seedlings were grown in a basic nutrient solution with either 0.1 mm (NH₄)₂SO₄ and 0.1 mm KNO₃ or 1 mm (NH₄)₂SO₄ and 1 mm KNO₃, with potassium concentrations adjusted, for 14 days. Subsequently, seedlings were transferred to fresh solution supplemented with 0.2/2 mm ¹⁵N‐NH₄Cl or 0.1/1 mm ¹⁵N‐ KNO₃ (98% ¹⁵N abundance; Sigma–Aldrich, Cat# 299251 to ¹⁵N‐NH₄Cl and Cat#335134 to ¹⁵N‐KNO₃) replace 0.1/1 mm (NH₄)₂SO₄ or 0.1/1 mm KNO₃ for 3 h. After labeling, roots were rinsed with 0.1 mm CaSO₄ for 1 min, separated from shoots, ground into powder, and analyzed for ¹⁵ N content using an isotope ratio mass spectrometer (Thermo Finnigan Delta Plus XP coupled with a Flash EA 1112 elemental analyzer). Each biological replicate included 20 seedlings (n = 4 biological replicates).

Measurement of Nitrogen Concentration

Rice seedlings were grown in 0.1 mm (NH₄)₂SO₄ and 0.1 mm KNO₃ or 1 mm (NH₄)₂SO₄ and 1 mm KNO₃ in basic nutrient solution for 14 days. The total N concentration was determined by the Kjeldahl method as described.^{[ 44 ]} Each biological replicate included 20 seedlings (n = 4 biological replicates).

Phosphorylation Assay in Rice Protoplast

To examine OsNLP3 phosphorylation under nitrate induction, nitrate‐free protoplasts stably expressing OsNLP3‐FLAG were treated with W5 solution containing 10 mm KNO₃ for 15 or 30 min, with or without 2 mm LaCl₃ pretreatment for 10 min. To test the effect of Ser193 on OsNLP3 phosphorylation, nitrate‐free protoplasts transfected with pCAMBIA2300‐35S‐OsNLP3‐FLAG, pCAMBIA2300‐35S‐OsNLP3^S193A‐FLAG, and pCAMBIA2300‐35S‐OsNLP3^S193D‐FLAG were incubated in W5 solution for 12 h, centrifuged at 200 × g, and treated with 10 mm KNO₃ for 30 min. To investigate the effects of OsNRT1.1B, OsCNGC14, and OsCNGC16 on OsNLP3 phosphorylation, nitrate‐free protoplasts of osnrt1.1b, oscngc14, and oscngc16 mutants transfected with pCAMBIA2300‐35S‐OsNLP3‐FLAG were incubated in W5 solution for 12 h, centrifuged at 200 × g, and treated with 10 mm KNO₃ for 30 min. Total proteins were extracted from lysed protoplasts using 50 µL extraction buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 1% Triton X‐100, 10% glycerol, 1× Protease and phosphatase inhibitor cocktail (NCM Biotech) at 4 °C for 30 min. Then treatment with or without λPP (Lambda Protein Phosphatase) (NEB) at 30 °C for 30 min, mixed with 2× SDS loading buffer at 100 °C for 10 min, and analyzed by SDS‐PAGE and Phos‐tag gels, immunoblotting using HRP‐conjugated anti‐FLAG. The X‐ray film was scanned and analyzed for statistics using ImageJ software.

Dual‐Luciferase Reporter Assay

Dual‐luciferase reporter assay was performed using nitrate‐free rice protoplasts. Protoplasts were isolated from 12‐day‐old seedlings and transformed as previously described.^{[ 42 ]} The pGreenII‐0800‐pNRT2.1‐LUC, pGreenII‐0800‐pNIA1‐LUC, pGreenII‐0800‐pNIR‐LUC, pGreenII‐0800‐pGS1.1‐LUC, and pGreenII‐0800‐pNLP3‐LUC vectors served as the reporters. The pCAMBIA2300‐35S‐OsNLP3‐FLAG, pCAMBIA2300‐35S‐OsNLP3^S193A‐FLAG, and pCAMBIA2300‐35S‐OsNLP3^S193D‐FLAG constructs described above were used as the effector. The empty pCAMBIA2300‐35S ‐FLAG vector served as the control. Protoplasts co‐expressing effector and reporters were incubated in W5 solution for 12 h. Then, 5 mm KNO₃ was used as a treatment, and 5 mm KCl was added as control for another 0–180 min of incubation. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured using a Dual Luciferase reporter assay kit (Promega, E1960) with a GloMax 20/20 luminometer (Promega). LUC/REN ratios were calculated to represent the relative LUC activity. For each plasmid combination, three independent transformations were performed.

Nitrate Induction Assay of Nitrate‐Responsive Genes by Different OsNLP3 Phospho‐Forms

Nitrate‐free osnlp3 protoplasts expressed with pCAMBIA2300‐35S‐OsNLP3‐FLAG, pCAMBIA2300‐35S‐OsNLP3^S193A‐FLAG, and pCAMBIA2300‐35S‐OsNLP3^S193D‐FLAG were incubated in W5 solution for 12 h. Then, 5 mm KNO₃ was added to the protoplasts as treatment, and 5 mm KCl was added as a control, followed by incubation for another 0–90 min. Protoplasts were collected by centrifuging at 200 × g for 3 min for gene expression analysis.

Statistical Analysis

All experiments were independently repeated in at least three biological replicates. Data are presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 10.4 software. For comparisons between two groups with normally distributed data, Student's t‐test was used. For comparisons among three or more groups, one‐way ANOVA followed by Tukey's test was employed. Statistical significance was defined as P < 0.05. Detailed statistical methods for each experiment are provided in the corresponding figure legends.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

X.W., Y.L., and W.L. contributed equally to this work. X.W., designed research, performed experiments, analyzed the data, and wrote the manuscript. W.L., Y.L., X.M., W.W., Z.J., and Y.W., conducted some of the experiments. Y.L., L.L., B.H., and C.C. designed research, wrote the manuscript and supervised the project.

Supporting information

Supporting Information

Wang X., Liu Y., Li W., et al. “OsNRT1.1B‐OsCNGC14/16‐Ca²⁺‐OsNLP3 Pathway: Phosphorylation‐Mediated Maintenance of Nitrogen Homeostasis.” Adv. Sci. 12, no. 43 (2025): e07919. 10.1002/advs.202507919

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.