Comparative Metabolomic and Transcriptomic Analysis of Flavonoid Accumulation in the Zest and Pith of Guang Foshou at Green and Ripe Stages

Citrus medica ‘Fingered’ (known as Guang Foshou in Chinese) is a distinctive edible fruit and a traditional herbal medicine enriched with bioactive flavonoids. However, the spatiotemporal patterns of flavonoid accumulation in the zest (flavedo) and pith (albedo) throughout fruit development remain poorly characterized. This study combined metabolomic and transcriptomic analyses to examine green and yellow fruits. Total flavonoid content increased significantly in the zest during fruit ripening, rising from 1.60 to 2.46 mg/g, whereas it remained relatively stable in the pith at approximately 2.2–2.3 mg/g. In the pith, flavanone glycosides, including hesperidin, neohesperidin, and naringin, accumulated progressively and are recognized for their cardioprotective and anti-inflammatory properties. In comparison, polymethoxylated flavones such as nobiletin and tangeretin, together with the methylated flavonol syringetin, accumulated in the zest, contributing to fruit coloration and showing well-known anti-inflammatory and anticancer activities. Transcriptomic analysis revealed that the flavonoid biosynthesis pathway involves core structural genes, including CHS, CHI, FLS, CYP75B2, FGT, and UFGT, which play crucial roles in regulating tissue-dependent accumulation patterns. Pathway analysis further showed that plant hormone signal transduction and carotenoid biosynthesis were enriched in the zest, with ethylene biosynthesis (ACO1) and abscisic acid biosynthesis (NCED2 and CCD4) genes upregulated during the green-to-yellow transition and co-expressed with flavonoid structural genes. We propose a two-tier system of quality markers for Guang Foshou: diosmin, which did not differ between the zest and the pith (q = 0.32) but declined by 22% from the green to the ripe stage (q = 0.0029), is suitable as a tissue-independent baseline marker provided that it is referenced to a defined maturity stage, whereas syringetin (67-fold higher in ripe zest than in ripe pith), 5-demethylnobiletin (43-fold) and the flavanone glycosides hesperidin, neohesperidin and naringin, which were strongly enriched in the pith, can discriminate tissue origin and ripening stage. This tiered framework provides a scientific basis for medicinal quality control and molecular breeding of Guang Foshou.