Age-dependent Epigenetic Control of Flavonoid Metabolism Underlies Chemical Defenses in Ancient Ginkgo Biloba

Ancient trees such as Ginkgo biloba exhibit remarkable longevity and sustained physiological vigor despite millennia of environmental stress, yet how aging reprograms their secondary metabolism and chemical defense remains unclear. Here, we investigated age-related epigenetic and metabolic changes across G. biloba individuals aged 1 to 1,070 yr. We identified DEFICIENS AGAMOUS-LIKE 1 (GbDAL1) as a central age-associated regulator of flavonol metabolism. GbDAL1 expression rises progressively with age but is significantly reduced in juvenilized branches; this shift is driven by reduced expression of the DNA methyltransferase chromomethylase2 (GbCMT2), leading to promoter hypomethylation. Restoring GbCMT2 activity reinstates promoter DNA methylation and suppresses GbDAL1 transcription. Functionally, GbDAL1 negatively regulates flavonol biosynthesis by directly repressing the flavonol synthase (GbFLS) gene and inhibiting the transcriptional activity of GbMYBF1, thereby attenuating expression of flavonol pathway genes. Although total flavonol biosynthesis declines with age, metabolite profiling revealed marked accumulation of methylated and prenylated flavonols in ancient trees, suggesting an adaptive metabolic shift toward more stable defensive metabolites. These metabolites, together with diverse terpenoids, phenols and alkaloids, accumulate in the heartwood to form a persistent chemical barrier that supports long-term defense. Collectively, our findings reveal an epigenetically mediated age-metabolite regulatory axis in G. biloba, uncovering a molecular mechanism that links aging to secondary metabolic reprogramming and may contribute to the exceptional longevity and resilience of perennial plants.