High concentrations of CPPU promotes cucurbitacin B accumulation in melon (Cucumis melo var. makuwa Makino) fruit by inducing transcription factor CmBt.

N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU) is a cytokinin-like plant growth regulator, which application in melon fruit set often produced bitter fruit caused by cucurbitacin B (Cu B) accumulation. However, more evidence is required to uncover the role of CPPU in regulating Cu B synthesis. In this study, two oriental melon cultivars 'YMR' (easy to present bitter fruit in maturation) and 'HDB' (hardly produce bitter fruit at maturity) were used. Four concentrations of CPPU (2.5, 5, 10 and 20 mg L-1) were set and hand pollination was used as control. Cu B accumulated in roots and fruit of 7 days after flower (7 DAF), which in 'YMR' was higher than those in 'HDB', and consistent with Cu B biosynthetic genes expression patterns. Furthermore, Cu B content in fruit significantly increased with CPPU concentrations and reached the highest level at 7 DAF, then decreased after 14 DAF, and which treated by 20 mg L-1 CPPU was always higher than that of controls and other low CPPU concentrations. Meanwhile, fruit bitterness evaluation suggested 20 mg L-1 CPPU increased the occurrence of bitterness during melon maturation. Transcription analysis suggested that the expression of Cu B biosynthetic genes (CmBi, Cm710, CmACT) and CmBt, the fruit-specific transcription factor, were significantly induced by 20 mg L-1 CPPU. Transient over-expression of CmBt in young fruit of 'YMR' increased Cu B biosynthetic genes (CmBi, CmACT, Cm710 and Cm890) expression and promoted Cu B accumulation. Taken together, this study demonstrates that 20 mg L-1 CPPU promotes Cu B accumulation in melon fruit by inducing CmBt and its biosynthetic genes.