LAUSR

Tree peony ( Paeonia suffruticosa Andr.) is a popular ornamental plant in China due to its showy and colorful flowers. However, yellow-colored flowers are rare in both wild species and domesticated cultivars. The molecular mechanisms underlying yellow pigmentation remain poorly understood. Here, petal tissues of two tree peony cultivars, “High Noon” (yellow flowers) and “Roufurong” (purple–red flowers), were sampled at five developmental stages (S1–S5) from early flower buds to full blooms. Five petal color indices (brightness, redness, yellowness, chroma, and hue angle) and the contents of ten different flavonoids were determined. Compared to “Roufurong,” which accumulated abundant anthocyanins at S3–S5, the yellow-colored “High Noon” displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols but no anthocyanin production. The contents of THC, flavones, and flavonols in “High Noon” peaked at S3 and dropped gradually as the flower bloomed, consistent with the color index patterns. Furthermore, RNA-seq analyses at S3 showed that structural genes such as PsC4H s, PsDFR s, and PsUFGT s in the flavonoid biosynthesis pathway were downregulated in “High Noon,” whereas most PsFLS s, PsF3H s, and PsF3’H s were upregulated. Five transcription factor (TF) genes related to flavonoid biosynthesis were also upregulated in “High Noon.” One of these TFs, PsMYB111, was overexpressed in tobacco, which led to increased flavonols but decreased anthocyanins. Dual-luciferase assays further confirmed that PsMYB111 upregulated PsFLS . These results improve our understanding of yellow pigmentation in tree peony and provide a guide for future molecular-assisted breeding experiments in tree peony with novel flower colors.