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Flavonols and proanthocyanidins (PAs) are the main pigments in the black spines of cucumber (Cucumis sativus) fruit, and CsMYB60 is a key regulator of the biosynthesis of flavonols and PAs. However, in cucumber, the tissue distribution pattern of flavonols and PAs and the mechanism of their biosynthesis regulated by CsMYB60 remain unclear. In this study, we clarified the tissue-specific distribution of flavonoids and the unique transcriptional regulation of flavonoid biosynthesis in cucumber. CsMYB60 activated CsFLS and CsLAR by bindin