Transcriptomics and Metabolomics Integrated Analysis Provide Insights Into the Differential Accumulation of Bitterness in Pummelo ( <i>Citrus grandis</i> )

Natural flavonoids give citrus its unique color and flavor, and directly affect its bitterness. However, the variations in bitterness and the underlying mechanisms among different citrus varieties remain poorly understood. In this study, three pummelo varieties with distinct bitter flavor profiles were selected for metabolomic and transcriptomic analyses to investigate the accumulation of bitter compounds in citrus. A total of 439 reliable metabolites were identified, with pathway analysis revealing significant enrichment of "Flavone and flavonol biosynthesis" and "Flavonoid biosynthesis" pathways in both bitter and non-bitter pummelo varieties. Comparative analysis identified 18 differentially expressed flavonoids, and HPLC analysis confirmed that naringin and neohesperidin are the primary contributors to bitterness. All 8658 differentially expressed genes (DEGs) were clustered into eight groups using K-means analysis. Cluster 5 exhibited an expression trend consistent with the bitter taste accumulation pattern across the three pummelo varieties. Pathway enrichment analysis indicated that DEGs in cluster 5 were significantly associated with the phenylalanine metabolic pathway. Using the enhanced 'GFAnno' flavonoid gene annotation tool, four 7-Glct, five 1,2-Rhat, and five 1,6-Rhat flavone-related genes, along with phenylalanine-related genes, were identified. Expression analysis and RT-qPCR results demonstrated that key genes PAL, C4H, and 4CL in the upstream phenylpropanoid pathway of flavonoid biosynthesis were highly expressed in the two bitter pummelo cultivars, but low in the non-bitter pummelo. Joint analysis further revealed that the transcription factors MUTE/bHLH (XGF149920) and NID1/MYB-like (XGF105200) were co-expressed with naringin and neohesperidin, suggesting their involvement in regulating bitter flavanone metabolism.