PbbHLH137 interacts with PbGIF1 to regulate pear fruit development by promoting cell expansion to increase fruit size.

The regulation of fruit development is a complex process and a core issue in the fruit tree industry. To investigate the role of PbGIF1 in pear fruit development, we identified a transcription factor PbbHLH137 that regulates pear (Pyrus bretschneideri) fruit development by screening a yeast library constructed from fruit cDNA. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and split luciferase complementation (split-LUC) assays were performed to confirm the PbbHLH137-PbGIF1 interaction. By tracing the complete fruit development process, we found that PbbHLH137 expression was closely related to fruit size and highly involved at the late pear fruit development stage. Transgenic experiments showed that heterologous expression of PbbHLH137 or PbGIF1 promoted fruit enlargement. PbbHLH137 promoted mainly the expansion of fruit cell volume, whereas PbGIF1 mainly increased the number of cells. Further LUC experiments demonstrated that PbGIF1 promoted the transcriptional activation ability of PbbHLH137. Our work identified PbbHLH137 as a transcription factor that regulates fruit development, and showed that PbGIF1 played an ongoing role during fruit development, making it a candidate gene for genetic improvement of pear fruit development.

PbXND1 Results in a Xylem-Deficient Dwarf Phenotype through Interaction with PbTCP4 in Pear (Pyrus bretschneideri Rehd.).

Dwarfing is an important agronomic characteristic in fruit breeding. However, due to the lack of dwarf cultivars and dwarf stocks, the dwarfing mechanism is poorly understood in pears. In this research, we discovered that the dwarf hybrid seedlings of pear (Pyrus bretschneideri Rehd.), 'Red Zaosu,' exhibited a xylem-deficient dwarf phenotype. The expression level of PbXND1, a suppressor of xylem development, was markedly enhanced in dwarf hybrid seedlings and its overexpression in pear results in a xylem-deficient dwarf phenotype. To further dissect the mechanism of PbXND1, PbTCP4 was isolated as a PbXND1 interaction protein through the pear yeast library. Root transformation experiments showed that PbTCP4 promotes root xylem development. Dual-luciferase assays showed that PbXND1 interactions with PbTCP4 suppressed the function of PbTCP4. PbXND1 expression resulted in a small amount of PbTCP4 sequestration in the cytoplasm and thereby prevented it from activating the gene expression, as assessed by bimolecular fluorescence complementation and co-location analyses. Additionally, PbXND1 affected the DNA-binding ability of PbTCP4, as determined by utilizing an electrophoretic mobility shift assay. These results suggest that PbXND1 regulates the function of PbTCP4 principally by affecting the DNA-binding ability of PbTCP4, whereas the cytoplasmic sequestration of PbTCP4 is only a minor factor. Taken together, this study provides new theoretical support for the extreme dwarfism associated with the absence of xylem caused by PbXND1, and it has significant reference value for the breeding of dwarf varieties and dwarf rootstocks of the pear.

PbWRKY75 promotes anthocyanin synthesis by activating PbDFR, PbUFGT, and PbMYB10b in pear.

Anthocyanins are common secondary metabolites in plants that impart red coloration to fruits and flowers. The important WRKY transcription factor family plays multifaceted roles in plant growth and development. In this study, we found a WRKY family gene, Pyrus bretschneideri WRKY75, that may be involved in anthocyanin synthesis in pear. Unlike Arabidopsis thaliana WRKY75, PbWRKY75 may be a positive regulator of anthocyanin synthesis. A transient expression assay indicated that PbWRKY75 promoted pear anthocyanin synthesis. The structural genes (PbANS, PbDFR, and PbUFGT) and positive regulators (PbMYB10 and PbMYB10b) of anthocyanin synthesis were significantly upregulated in the fruitlet skins of PbWRKY75-overexpressing "Zaosu" pears. Subsequently, yeast one-hybrid and dual-luciferase assays indicated that PbWRKY75 promoted PbDFR, PbUFGT, and PbMYB10b expression by activating their promoters. These results revealed that PbWRKY75 may promote the expression of both PbMYB10b and anthocyanin late biosynthetic genes (PbDFR and PbUFGT) by activating their promoters, thereby inducing anthocyanin synthesis in pear. This study enhanced our understanding of the mechanism of pear anthocyanin synthesis, which will be beneficial in the improvement of pear peel color.