Parthenocarpy can ensure fruit setting without fertilization and generate seedless fruits. PbCYP78A6 has been shown to play a role in gibberellin (GA)-induced parthenocarpy in pears. However, the transcriptional response mechanism of PbCYP78A6 to GA remains unclear. In this study, using a yeast one-hybrid assay combined with co-expression analysis, PbMYB56 was initially identified as a transcription regulator of PbCYP78A6, which was further confirmed by electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assays. The biofunction of PbMYB56 was further verified using transient transgene tests, stable transgenic pear callus and tomato. PbMYB56 overexpression resulted in reduced cell death and higher fluorescence intensity after fluoresce diacetate (FDA) staining, as well as delayed fruit-drop by increasing PbCYP78A6 expression in unpollinated pear fruitlets and callus. In contrast, silencing PbMYB56 caused cell death and early fruit-drop with decreased PbCYP78A6 expression. Moreover, after emasculation, heterologous overexpression of PbMYB56 induced parthenocarpy and enlarged seed size in pollinated tomato fruits. Silencing SlMYB56, a homolog of PbMYB56 in tomatoes, resulted in smaller fruit and seed size, and these traits were restored by co-overexpression with PbCYP78A6. Furthermore, we investigated the protein interaction between PbMYB56 and PbDELLA, which is crucial component of the GA signaling pathway. This interaction inhibited PbMYB56-induced transcriptional activation of PbCYP78A6. Co-overexpression of PbMYB56 and PbDELLA contributed to reduced seed development and loss of parthenocarpy potential in tomatoes. Collectively, our study identifies PbDELLA-PbMYB56-PbCYP78A6 as a regulatory module of GA_4 + 7-induced pseudo-embryo and parthenocarpy development, offering insights into the mechanism underlying parthenocarpy formation in pears.

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