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

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.