Autotetraploidization Induces a Metabolic Shift from Flavonoids to Coumarins While Maintaining Volatile Stability in Yuzu (Citrus junos Sieb. ex Tanaka)

Polyploidy modifies metabolic profiles and transcriptional regulation of biosynthetic pathways. Citrus tetraploids are characterized by dwarf growth and increased leaf biomass. Citrus leaves are valuable resources for essential oils and natural food additives because of their rapid regrowth, high biomass yield, and year-round availability. In this study, 11 spontaneous autotetraploids (1.14%) were identified among 967 yuzu seedlings. Compared with diploids, tetraploids exhibited reduced plant height, wider leaves, and fewer but larger stomata, accompanied by a 70% increase in net photosynthetic rate and a 2.6-fold increase in stomatal conductance. Volatilomic analysis showed that only 12.4% of the 920 detected volatile organic compounds (VOCs) differed significantly between ploidy levels; notably, two esters—methyl 2-(methylamino) benzoate and 2-methoxyethyl benzoate—were substantially enriched in tetraploids (~400-fold and ~8-fold, respectively). Nonvolatile metabolomic analysis revealed higher accumulation of bioactive coumarins (e.g., bergapten, imperatorin, and isopimpinellin) and lower levels of flavonoids in tetraploid leaves. Transcriptomic analysis indicated enrichment of genes involved in flavonoid and coumarin biosynthesis. Integrated multi-omics analysis demonstrated that upregulation of psoralen synthase (PS) and scopoletin 8-hydroxylase (S8H) was positively associated with increased coumarin accumulation, whereas downregulation of flavonol synthase (FLS) and flavonol-3-O-glucoside L-rhamnosyltransferase (FG2) contributed to reduced flavonoid content, indicating a metabolic shift from flavonoids to coumarins in tetraploid leaves. These findings provide insight into secondary metabolite reprogramming following autotetraploidization in yuzu and highlight its potential value for the bioactive coumarin industry.