OsCIPK18 Mediates Nitrate-Regulated Rice Root Growth through the Nitrogen Metabolism Pathway: Insights from Quantitative Proteomic and Phosphoproteomic Analyses

Nitrate acts as both a nutrient and a signaling molecule to regulate root growth, and this process is closely associated with protein abundance and protein phosphorylation within the nitrogen metabolism pathway. However, the relationship between nitrate-regulated root growth, protein expression and protein phosphorylation remain incompletely understood. Here, we investigated the function and underlying molecular mechanisms of the calcineurin B-like (CBL)-interacting protein kinase OsCIPK18 in nitrate-modulated rice root growth using phenotypic analyses together with quantitative proteomic and phosphoproteomic profiling of wild-type (WT) plants and cipk18 mutants. Knockdown of OsCIPK18 significantly inhibited rice root growth compared with WT plants. In contrast, 2 mM nitrate significantly promoted root growth in the cipk18 mutant, increasing lateral root length by 65% and radicle length by 24%, whereas these effects were not observed in WT plants. Consistently, knockdown of OsCIPK18 altered the accumulation of nitrogen-related proteins (including GS1;2, OsGS2, OsNADH-GOGAT2 and OsbetaCA2) and the phosphorylation status of the high-affinity nitrate transporter OsNRT2.2 in response to nitrate. Together, these findings reveal a central regulatory role of OsCIPK18 in nitrogen signaling and root development and provide a potential molecular target and theoretical basis for breeding rice varieties with improved nitrogen use efficiency. • OsCIPK18 interacts with OsCBL1 to mediate nitrate-regulated root growth in rice. • The nitrate-responsive phosphoproteome is dependent on OsCIPK18. • Nitrate-induced accumulation of GS1;2, OsGS2 and OsNADH-GOGAT2 requires OsCIPK18. • OsNRT2.2 phosphorylation at S57/T524/S529 is nitrate- and OsCIPK18-dependent. • OsCIPK18 fine-tunes nitrate signaling via OsCRL4 kinase and OsPP18 phosphatase.