PbBPC4 involved in a xylem-deficient dwarf phenotype in pear by directly regulating the expression of PbXND1.

Dwarfing is an important agronomic trait in fruit breeding. At present, dwarf cultivars or dwarfing rootstocks are used for high-density planting. Although some dwarf rootstocks have been used in the cultivation of pear (Pyrus bretschneideri Rehd), the breeding of dwarf pear rootstocks or cultivars is still sorely lacking. A previous study reported that PbXND1 results in a xylem-dwarf phenotype in pear trees. However, the regulatory mechanism upstream of PbXND1 is unclear. In this study, we identified PbBPC4 as an upstream regulatory factor of PbXND1 in yeast one-hybrid assays. In ß-glucuronidase staining and dual-luciferase assays, PbBPC4 enhanced the activity of the PbXND1 promoter. Tobacco plants overexpressing PbBPC4 showed decreased plant height because of a reduced xylem size. Similar changes in the xylem was observed in transgenic pear roots; those overexpressing PbBPC4 showed reduced xylem size, and those with silencing PbBPC4 expression showed increased xylem size, greater density of xylem vessels, and a larger proportion of the xylem out of the total cross-section area. Expression analyses showed that PbBPC4 increases the transcription of PbXND1, leading to reduced transcript levels of genes involved in the positive regulation of xylem development, ultimately resulting in a xylem-deficient dwarf phenotype. Taken together, our results reveal the mechanism by which PbBPC4 participates in the regulation of xylem development via directly altering the expression of PbXND1, thus leading to the dwarf phenotype in pear. These findings have reference value for the breeding of dwarf pear trees.

PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3.

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars ('Cuiguan', 'Hongsucui') and two P. ussuriensis cultivars ('Qiuxiang', 'Hanhong'), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.

'Yunnan' quince rootstock promoted flower bud formation of 'Abbé Fetel' pear by altering hormone levels and PbAGL9 expression.

Flower busd formation is an important plant growth process. It has been reported that dwarfing rootstocks can significantly affect the flower bud formation of scions. In this study, we found the dwarfing rootstock 'Yunnan' quince could significantly increase the flowering rate of 'Abbé Fetel' pear scions. The RNA-sequencing data revealed significant changes in the expression of genes related to hormone pathways. Furthermore, hormone analyses indicated that 'Yunnan' quince significantly decreased the GA3 content and increased the cytokinin/auxin ratio in 'Abbé Fetel' pear apical buds. The hormone contents were consistent with the RNA-sequencing data. Moreover, we found the flower development-related genes PbAGL9 and PbCAL-A1 were significantly upregulated and PbTFL1 was significantly downregulated in 'Abbé Fetel'/'Yunnan' quince apical buds. To further clarify the relationship between hormones and flowering-related genes, a hormone response assay was carried out. We found the expression levels of PbCAl-A1, PbTFL1 and PbAGL9 were regulated by hormones including GA3, CPPU and NAA. Y1H and dual-luciferase assays indicated that PbAGL9 significantly decreased the promoter activity of PbTFL1. In summary, 'Yunnan' quince upregulated PbCAL-A1 and PbAGL9, and downregulated PbTFL1 expression by decreasing the GA3 content and increasing the cytokinin/auxin ratio in 'Abbé Fetel' pear apical buds. Additionally, 'Yunnan' quince down-regulate PbTFL1 by upregulating the expression of PbAGL9, and eventually promoted floral induction in 'Abbé Fetel' pear.