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.

In most fleshy fruits, including apple, cherry, plum, and grape, citric acid content declines during the late stages of fruit development, a process that largely determines fruit flavor. In contrast, pear (Pyrus spp.) exhibits a less common developmental pattern characterized by continued citric acid accumulation during late fruit development. Although similar patterns have been reported in a limited number of other fruit species and germplasms, the molecular mechanisms underlying this developmental trajectory remain largely unknown. Here, comparative analyses of organic acid dynamics among cultivated pear varieties revealed that citric acid predominantly accumulates during late fruit development. Transcriptome analysis combined with M-fuzz soft clustering identified the vacuolar dicarboxylate transporter PutDT and the NAC transcription factor PuNAC2 as candidate genes closely associated with this developmental pattern. Electrophysiological analyses demonstrated that PutDT functions as a pH-dependent vacuolar transporter for both citrate and malate, with a higher affinity for citrate under acidic conditions. Functional analyses further demonstrated that PutDT promotes citric acid accumulation in pear fruit, callus, and a heterologous tomato system. Furthermore, PuNAC2 directly activated PutDT transcription by binding to the SNBE motif in its promoter, and its positive effect on citric acid accumulation depended on PutDT activation. Collectively, these findings reveal a PuNAC2–PutDT regulatory module controlling vacuolar citric acid accumulation during pear fruit development and provide molecular insights into the regulation of fruit acidity, as well as potential molecular targets for acidity improvement.