Dynamic Profiling of Polymethoxyflavones and Biosynthetic Insights in the Peel of Citrus depressa During Fruit Development

Background/Objectives: The peel of Citrus depressa Hayata is exceptionally rich in polymethoxyflavones (PMFs), particularly nobiletin, which exhibits diverse bioactivities. However, the developmental dynamics of PMF accumulation and its underlying molecular regulation remain poorly understood. This study aimed to characterize PMF profiles across peel development and to identify the key genes and critical stage governing nobiletin enrichment. Methods: Peel samples of C. depressa were collected at five developmental stages (S1–S5). Nobiletin content was quantified by high-performance liquid chromatography (HPLC). Widely targeted metabolomics was used to profile PMFs, and transcriptome sequencing combined with weighted gene co-expression network analysis (WGCNA) was performed to identify candidate structural genes. Expression patterns were validated by quantitative real-time PCR (qRT–PCR). Results: Nobiletin showed a bimodal accumulation pattern, peaking at stages S1 and S4 (4.637 mg/g fresh weight at S4). Metabolomics identified 68 PMFs, among which nobiletin was the dominant component (44.5–51.5% of the total PMF peak-area signal). Transcriptome and WGCNA analyses revealed that OMT, CYP450, and UGT genes were co-upregulated at S4, with qRT–PCR confirming expression peaks coinciding with nobiletin accumulation. Conclusions: Nobiletin accumulates in a bimodal pattern in C. depressa peel, peaking at S4 (4.637 mg/g FW), which is identified as the optimal harvest window. The coordinated upregulation of O-methyltransferase (OMT), cytochrome P450 (CYP450), and UDP-glycosyltransferase (UGT) genes at S4 provides candidate targets for the directed utilization and molecular breeding of C. depressa.