Sweet cherry AP2/ERF transcription factor, PavRAV2, negatively modulates fruit size by directly repressing PavKLUH expression

For sweet cherry, fruit size is one of the main targets in breeding programs owing to the high market value of larger fruits. KLUH/CYP78A5 is an important regulator of seed/fruit size in several plant species, but its molecular mechanism is largely unknown. In this study, we characterized the function of PavKLUH in the regulation of sweet cherry fruit size. The ectopic overexpression of PavKLUH in Arabidopsis increased the size of its siliques and seeds, whereas virus-induced gene silencing of PavKLUH in sweet cherry significantly decreased fruit size by restricting mesocarp cell expansion. We screened out an AP2/ERF transcription factor containing a B3-like domain, designated as PavRAV2, which was able to physically interact with PavKLUH promoter in a yeast one-hybrid (Y1H) system. In Y1H assays, electrophoretic mobility shift assays, and dual-luciferase reporter analyses, PavRAV2 directly bound to the promoter of PavKLUH in vitro and in vivo, and suppressed PavKLUH expression. Silencing of PavRAV2 resulted in enlarged fruit as a result of enhanced mesocarp cell expansion. Together, our results provide new insights into signaling pathways related to fruit size, and outline a possible mechanism for how the RAV transcription factor directly regulates CYP78A family members to influence fruit size and development.