**Background:** RWP-RKs are plant-specific transcription factors involved in nitrogen absorption and utilization, but their genome-wide identification and function in Compositae plants were largely unknown. Nitrogen plays a critical role in plant growth and flowering, yet the mechanisms linking RWP-RKs to flowering time regulation had not been explored in chrysanthemum.
**Methods:** The authors identified RWP-RK family members in Chrysanthemum lavandulifolium using BLAST (e-value < 1e-5) against the genome (GCA_022545495.1) with Arabidopsis RWP-RK sequences as references. Phylogenetic trees were constructed using maximum likelihood with 1000 bootstrap replicates. Conserved motifs, domains, gene structures, and promoter cis-elements were analyzed using MEME, NCBI CDD, and PlantCARE. Tissue-specific expression was assessed using transcriptome data (SRR14723013–SRR14723015, SRR14723028–SRR14723033) and validated by qRT-PCR with SAND as internal control. Plants were grown under controlled conditions (24±2°C, 3000 lx) with three nitrate treatments: LN (0.1 mM KNO3), ON (1.5 mM KNO3), and HN (3.0 mM KNO3). Flowering time was observed, and leaves at vegetative stage were collected after four treatments for gene expression analysis.
**Key Results:** A total of 101 RWP-RK genes were identified in C. lavandulifolium, including 38 NLPs, 31 RKDs, and 32 specific expansion members. For comparison, 56, 52, 41, and 27 RWP-RKs were found in Chrysanthemum nankingense, Helianthus annuus, Mikania micrantha, and Lactuca sativa, respectively, while only 14 were found in Arabidopsis thaliana. Of the 101 genes, 100 were mapped to 9 chromosomes, with chromosome 9 containing the most (39 genes). Tandem duplications were observed on chromosomes 1 and 9. Promoter analysis revealed light response elements, hormone response elements (MeJA, ABA, IAA, GA, salicylic acid), abiotic stress elements, N-responsive GCN4 elements in 17 genes, and circadian regulatory elements (CAAAGATATC) in 31 genes. In transcriptome data, 45 ClRWP-RK genes were detected in leaves and apical meristems, with 33 differentially expressed. qRT-PCR showed all 10 ClNLPs were highly expressed in apical meristem at floral transition stage; 7 ClNLPs (Cl34639.1, Cl04888.1, Cl60370.1, Cl06657.1, Cl20718.1, Cl61441.1, Cl17046.1) were up-regulated in leaves at floral transition. All 10 ClNLPs were negatively regulated by LN conditions; 3 (Cl04888.1, Cl60370.1, Cl62000.1) were induced by ON conditions. Flowering time was advanced under ON and delayed under LN and HN conditions. Under ON, ClCRYs, ClPHYC, ClGI, ClCOL4, ClCOL5, ClFT, ClFLC, and ClSOC1 were up-regulated, while ClTFL was down-regulated. ClCRY1a, ClCRY1c, ClCRY2a, and ClCRY2c expression under ON at vegetative stage reached levels comparable to short-day induction at reproductive stage.
**Clinical Implications:** This study provides the first genome-wide characterization of RWP-RK genes in chrysanthemum and establishes a mechanistic link between nitrate signaling and flowering time regulation. The finding that optimal nitrate advances flowering by up-regulating cryptochrome genes has practical implications for ornamental horticulture, potentially enabling manipulation of flowering time through nitrogen management. The identification of ClNLPs as candidate regulators connecting nitrogen status to photoperiodic flowering offers new targets for breeding programs aimed at improving nitrogen use efficiency and flowering control in chrysanthemum and related Compositae species.