Nitrate (NO3‒) serves as both a major nitrogen (N) source for most plants and a key signalling molecule regulating plant growth and development 1. In rice, nitrate signalling is initiated via the OsNRT1.1B–OsSPX4–OsNLP3 cascade 2. Calcium (Ca2+) signalling, which acts as a central second messenger in plants, responses to a wide range of internal developmental cues and external environmental stimuli 3. Although it has been suggested that nitrate-triggered Ca2+ activation is essential for initiating the primary nitrate response (PNR) in plants 4, the mechanism generating this nitrate-specific Ca2+ signature remained unclear in rice. Moreover, the coordination between the rapid PNR and long-term N utilization has been poorly understood. Recently, Wang et al. (2025) identified a novel nitrate signalling pathway in rice, the OsNRT1.1B/OsCNGC–Ca2+–OsNLP3 cascade, which is in parallel with the established ubiquitination-mediated OsNRT1.1B–OsSPX4–OsNLP3 axis. Their work elucidates how Ca2+ signal regulates nitrate responses (Figure 1) and bridges a gap between short-term PNR and long-term N utilization through integrating the phosphorylation and ubiquitination pathways, consequently proposing a more unified model of N homoeostasis 5. A Dual-pathway for nitrate-responsive involving in calcium signalling and ubiquitination cascade mediated by OsNRT1.1B in rice. (1) OsNRT1.1B-OsCNGC14/16-Ca2+ pathway: the OsCNGC14/16-mediated Ca2+ influx leads to OsNLP3 phosphorylation at Ser193, facilitating its nuclear import and autoactivation. (2) OsNRT1.1B-OsNBIP1-OsSPX4 pathway: the OsNBIP1-mediated ubiquitination and degradation of the inhibitory protein OsSPX4 liberates OsNLP3, ensuring prolonged transcriptional programming for long-term nitrogen utilization. In Arabidopsis, the dual-function transceptor AtNRT1.1 facilitates nitrate-induced Ca2+ influx 6. To explore the role of Ca2+ signalling in rice and its dependence on OsNRT1.1B, the closest orthologue of AtNRT1.1, Wang et al. (2025) profiled the expression of cyclic nucleotide-gated channel (CNGC) family genes encoding putative Ca2+ channels. They identified two nitrate-responsive OsCNGC members, OsCNGC14 and OsCNGC16, both are essential for triggering the nitrate-mediated Ca2+ influx in rice. Knockout of either OsCNGC14 or OsCNGC16 abolished Ca2+ signalling and suppressed PNR. Interestingly, co-expression of OsCNGC14 and OsCNGC16—but neither alone—produced robust Ca2+ currents in Xenopus oocytes, indicating that functional channel activity requires heterodimer assembly. These results contrast with the observations in Arabidopsis, in which AtCNGC15 alone can elicit calcium signals 6. Notably, OsCNGC14 and OsCNGC16 form a plasma membrane-localized complex with OsNRT1.1B in root tips. Co-expression of OsNRT1.1B with OsCNGC14/16 in Xenopus oocytes suppressed Ca2+ currents, indicating that OsNRT1.1B inhibits the channel complex. Nitrate application, however, could alleviate this inhibition and activate the channel and triggering Ca2+ influx. This Ca2+ signal ultimately induces phosphorylation of OsNLP3-a central transcription factor in nitrate signalling, thereby promoting its nuclear translocation and enhancing its transcriptional activity (Figure 1). Crops have evolved multiple regulatory pathways for nutrient acquisition and utilization to adapt to dynamic environmental conditions. Rice, as most terrestrial plants, can operate a highly-efficient mycorrhizal uptake pathway via association with arbuscular mycorrhizal (AM) fungi, in addition to the direct root uptake pathway, to enhance the acquisition of certain nutrients, particularly phosphorus (P) and N, from soil 7. A recent study demonstrated that the OsNLP3–OsSPX4–OsPHR2 module can coordinate both direct and mycorrhizal nitrate uptake pathways via regulating the OsNAR2.1–OsNRT2s transporter complexes. The AM symbiosis upregulates OsNAR2.1–OsNRT2s, OsNLP3, OsPHR2, and OsSPX4 in arbuscule-containing cells, while attenuates their expression in epidermal cells, thereby fine-tuning N and P absorption at the symbiotic interface 8. Such a flexible switching between dual-uptake strategies enables plants to better adapt to the largely fluctuated nutrient availability in the complex rhizospheric environment. The dual role of the OsNLP3-OsSPX4-OsPHR2 ubiquitination degradation pathway in mediating both direct and symbiotic N-P signalling has provided significant inspiration, suggesting that the calcium signalling-induced phosphorylation pathway may play a potentially crucial role in mycorrhizal symbiosis or mycorrhizal nitrate uptake. Beyond nitrate signalling, CNGC-generated nuclear Ca2+ oscillations are also implicated in various biotic and abiotic stress responses—including symbiotic signalling, environmental adaptation, and hormone transduction 9. In Medicago truncatula, MtCNGC15 is essential for both Nod factor– and Myc factor–induced nuclear Ca2+ spiking; the mutation of MtCNGC15 impaired nodulation and mycorrhizal colonization 3, highlighting the crucial roles of CNGC proteins in symbiotic Ca2+ signalling. In rice, OsCNGC4/8/15/16 and OsNRT1.1B showed an upregulated expression in mycorrhizal roots 7. Since the OsNLP3–OsNAR2.1 module coordinates both direct and mycorrhizal nitrate uptake pathways, we hypothesize that a OsCNGC–OsNRT1.1B module might be able to mediate nitrate-induced Ca2+ influx, leading to OsNLP3 phosphorylation and thereby regulating AM symbiosis and mycorrhizal nitrate acquisition. Additionally, it remains to be further investigated whether calcium oscillations regulate symbiotic and nitrate signalling through the same or distinct CNGCs. Elucidating this issue is crucial for future research into the integration of crosstalk among calcium, nitrate, and symbiotic signalling pathways. Intriguingly, the OsCNGC14/16 proteins are also involved in abscisic acid (ABA) and stress signalling pathways, including responses to temperature fluctuations, chilling, and drought 9, which establishes a direct molecular link between nutrient status and stress resilience. Under nitrate-sufficient condition, activation of the OsNRT1.1B-OsCNGC14/16 pathway may prime the Ca2+ signalling network, potentially enhancing the plant's capacity to respond to subsequent stress signals that also involve Ca2+. Conversely, under stress condition such as drought, which engages ABA signalling, resource allocation may be shifted away from N acquisition and growth. It is plausible that the ABA signalling pathway could potentially suppress nitrate-triggered Ca2+ influx, thereby inhibiting growth under unfavourable conditions. The dual activation mechanism of OsNLP3 via phosphorylation at Ser193 illustrates a sophisticated evolutionary adaptation in rice. In Arabidopsis, AtNLP7 activation strictly relies on the phosphorylation by CPKs 4. By contrast, in rice, phosphorylation acts as an accelerator rather than a strict on/off switch: a phosphomimetic (S193D) variant of OsNLP3 enhances its nuclear translocation and transactivation, while a phosphoablative (S193A) variant retains partial activity. These findings suggest that the OsSPX4 ubiquitination-mediated release of OsNLP3 provides essential baseline activation, whereas Ca2+-dependent phosphorylation delivers a rapid boost 5. This layered regulatory strategy likely underpins rice's adaptability to fluctuating N supplies in paddy field 10. Despite these insights, the identity of the kinase phosphorylating OsNLP3 downstream of Ca2+ signalling remains unknown. In conclusion, the work by Wang et al. (2025) reframes nitrate signalling in rice from a linear cascade into an integrated dual-input control system. The identification of the OsNRT1.1B–OsCNGC14/16–OsNLP3 pathway offers an alternative avenue for developing next-generation crops with enhanced N use efficiency (NUE) and resilience, particularly under adverse environmental conditions. Key strategies include the precision modification of signalling components—such as generating phosphomimic OsNLP3 variants or engineering OsNRT1.1B variants with enhanced affinity for OsCNGCs—to boost plant responsiveness under low-nitrate conditions. These strategies advance beyond conventional approaches, which are primarily limited to the identification and utilization of natural favourable alleles or overexpression of genes. The ultimate goal is to develop 'smart' crops capable of sensing local and systematic nitrate levels, amplifying signals through Ca2+-mediated phosphorylation, and executing spatially precise adaptive responses in root architecture and N utilization. Such innovations could reduce reliance on synthetic fertilizers and contribute to a more sustainable and efficient agricultural system in the future. Shuangshuang Wang: conceptualization, visualization, writing – original draft. Guohua Xu: conceptualization, writing – review and editing. This work was supported by National Natural Science Foundation of China (Grant 32302665). The authors declare no conflicts of interest. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
Wang et al. (2025) studied this question.