Transcriptomic Analysis Reveals the Molecular Mechanisms Underlying Heat-Induced Suppression of Polymethoxyflavone Accumulation in Citrus Leaves

High-temperature stress impairs plant growth and alters secondary metabolism. Polymethoxyflavones (PMFs) are citrus-specific flavonoids with important nutritional benefits; however, their transcriptional responses to heat stress remain poorly understood. Here, five-month-old ‘Ponkan’ citrus seedlings were exposed to 40 °C for 6, 11, and 21 days. HPLC analysis showed that the accumulation of four major PMFs (sinensetin, nobiletin, tangeretin, and 5-demethylnobiletin) was significantly reduced in leaves under heat stress. RNA-seq identified 3424 differentially expressed genes shared across all three time points, which were enriched in pathways associated with microtubule cytoskeleton organization, cell cycle regulation, and glyoxylate and dicarboxylate metabolism. Further analysis of the PMF biosynthetic pathway revealed that 14 of 18 key structural genes, including CHS, CHI, FNSII, and OMT family members, were downregulated by heat treatment. In addition, several bHLH, AP2/EREBP, and MYB transcription factors, known regulators of flavonoid biosynthesis, exhibited expression patterns closely associated with PMF accumulation. RT-qPCR analysis validated the transcriptome results. Collectively, these findings suggest that heat stress suppresses PMF accumulation through coordinated repression of PMF biosynthetic genes and their potential regulators. This study provides new insights into the molecular basis of heat-responsive PMF metabolism and offers potential targets for maintaining citrus nutritional quality under elevated temperatures.