Pear fruit corking disorder is a non-infectious physiological condition that primarily occurs during the late developmental stages of pear and significantly impacts fruit quality and economic value in several major cultivars. As the underlying mechanism remains unclear, effective prevention strategies and genetic improvements continue to present major challenges. In this study, ‘Akizuki’ pear was used as experimental material, and the lignin content in cork-affected tissue was found to be significantly higher than in healthy tissue. Exogenous abscisic acid (ABA) treatment induced cork formation and promoted lignin biosynthesis, a major structural component, in both pear fruits and calli. Through integrated RNA-seq and expression analyses, we identified the ABA-responsive gene PbrMYB8, which was differentially expressed between healthy and diseased tissues and associated with lignin biosynthesis during corking disorder. Stable transformation of PbrMYB8 into pear calli and Arabidopsis confirmed its role in promoting lignin biosynthesis. Notably, PbrMYB8 not only activated lignin biosynthesis genes independently but also interacted with PbrMYB169 to form a protein complex that co-regulated their expression. These findings improve our understanding of lignin biosynthesis in pear fruit corking disorder by identifying a key regulator and its interaction network and provide a theoretical foundation for future strategies aimed at improving pear fruit quality.

Pyrus pyrifolia, commonly known as sand pear, is a key economic fruit tree in temperate regions that possesses highly diverse germplasm resources for pear quality improvement. However, research on the relationship between resistance and fruit quality traits in the breeding of fruit species like pear is limited. Pan-transcriptomes effectively capture genetic information from coding regions and reflect variations in gene expression between individuals. Here, we constructed a pan-transcriptome based on 506 samples from different tissues of sand pear, and explored the intrinsic relationships among phenotypes and the selection for disease resistance during improvement based on expression presence/absence variations (ePAVs). The pan-transcriptome in this study contains 156,744 transcripts, among which the novel transcripts showed significant enrichment in the defense response. Interestingly, disease resistance genes are highly expressed in landraces of pear but have been selected against during the improvement of this perennial tree species. We found that the genetically diverse landraces can be divided into two subgroups and inferred that they have undergone different dispersal processes. Through co-expression network analysis, we confirmed that the formation of stone cells in pears, the synthesis of fruit anthocyanins, and the ability to resist stress are interrelated. They are jointly regulated by several modules, and the expression of regulatory genes has significant correlations with these three processes. Moreover, we identified candidate genes such as HKL1 that may affect sugar content and are missing from the reference genome. This study provides insights into the associations between complex fruit traits, while providing a database resource for pear disease resistance and fruit quality breeding.

The color of red-skinned pear (Pyrus spp.) is primarily attributed to accumulation of anthocyanins, which provide nutritional benefits for human health and are closely associated with the commercial value of fruits. Here, we reported the functional characterization of a R2R3-MYB repressor PyMYB107, which forms an ‘activator-repressor’ loop to control anthocyanin accumulation in the red-skinned pear. PyMYB107 overexpression inhibited anthocyanin biosynthesis in both pear calli and fruits, while virus-induced gene silencing of PyMYB107 increased anthocyanin accumulation in pear fruits. Furthermore, ectopic expression of PyMYB107 decreased anthocyanin accumulation in tomato, strawberry and tobacco. PyMYB107 can competitively bind to PybHLH3 with PyMYB10/MYB114, thereby suppressing the transcriptional activation of key anthocyanin biosynthesis genes, PyANS and PyUFGT. Site-directed mutagenesis showed that mutations within the R3 domain and EAR motif of PyMYB107 eliminated its repressive activity. Additionally, PyMYB107 exhibited a comparable expression pattern to PyMYB10/MYB114 and was transcriptionally activated by them. Our finding advanced comprehension of the repression mechanism underlying anthocyanin accumulation, providing valuable molecular insights into improving quality of pear fruits.

Pear (Pyrus bretschneideri) fruit stone cells are primarily composed of lignin and have strongly lignified cell walls. The presence of stone cells has a negative influence on fruit texture and taste, and thus the reduction of stone cell content in pear fruit is a key goal of breeding efforts. However, research into the key transcription factors and regulatory networks associated with pear fruit stone cell formation have been limited. We here used a combination of co-expression network and expression quantitative trait locus (eQTL) analyses in 206 pear cultivars with different stone cell contents to identify relevant genes; these analyses uncovered the gene PbrMYB4, a R2R3 MYB transcription factor gene. There was a strong positive correlation between relative PbrMYB4 expression levels in the fruit flesh and stone cell/lignin contents. Overexpression of PbrMYB4 significantly increased the lignin contents, whereas silencing of PbrMYB4 had the opposite effect, decreasing the contents of lignin. PbrMYB4 overexpression in pear calli significantly promoted lignin biosynthesis. In Arabidopsis thaliana, PbrMYB4 overexpression resulted in increasing lignin deposition, cell wall thickness of vessels and xylary fiber, and accelerating expression level of lignin biosynthetic genes. PbrMYB4 was found to activate 4-Coumarate:Coenzyme A Ligase (Pbr4CL1) by binding to AC-I elements in the promoter regions, as demonstrated with dual-luciferase reporter assays and a yeast one-hybrid assay. These results demonstrated that PbrMYB4 positively regulated lignin biosynthesis in pear fruit stone cells by activating lignin biosynthesis genes. This study improves our understanding of the gene regulatory networks associated with stone cell formation in pear fruit, providing guidance for molecular breeding of pear varieties with low stone cell content.

Genomic selection (GS) has the potential to improve selection efficiency and shorten the breeding cycle in fruit tree breeding. In this study, we evaluated the effect of prediction methods, marker density and the training population (TP) size on pear GS for improving its performance and reducing cost. We evaluated GS under two scenarios: (1) five-fold cross-validation in an interspecific pear family; (2) independent validation. Based on the cross-validation scheme, the prediction accuracy (PA) of eight fruit traits varied between 0.33 (fruit core vertical diameter) and 0.65 (stone cell content). Except for single fruit weight, a slightly better prediction accuracy (PA) was observed for the five parametrical methods compared with the two non-parametrical methods. In our TP of 310 individuals, 2 000 single nucleotide polymorphism (SNP) markers were sufficient to make reasonably accurate predictions. PAs for different traits increased by 18.21%–46.98% when the TP size increased from 50 to 100, but the increment was smaller (-4.13%–33.91%) when the TP size increased from 200 to 250. For independent validation, the PAs ranged from 0.11 to 0.45 using rrBLUP method. In summary, our results showed that the TP size and SNP numbers had a greater impact on the PA than prediction methods. Furthermore, relatedness among the training and validation sets, and the complexity of traits should be considered when designing a TP to predict the test panel.

Plant multidrug and toxic compound extrusion (MATE) genes play an important role in the process of detoxification, plant morphogenesis, and anthocyanin accumulation. However, whether the MATE gene family functions in pear peel coloration is still unknown. To evaluate and identify the MATE gene family members which are involving in anthocyanin accumulation and coloration in pear. In this study, 85 MATE genes were identified in the reference pear genome of ‘Dangshansuli’ through genome-wide identification. Based on gene structure and phylogenetic tree analysis, the MATE family was divided into five subfamilies. RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) indicated that the expression patterns of PbrMATEs were tissue-specific. 28.24% (24) of PbrMATE genes were expressed in the fruits, and 44.71% (38) of PbrMATE genes were expressed in the leaves. Additionally, we found that the expression levels of PbrMATE9, PbrMATE26, PbrMATE50, and PbrMATE69 in debagged fruits with red peel were significantly higher than those in bagged fruits without red peel, according to our bagging/debagging treatment of ‘Mantianhong’. The expression pattern of PbrMATE9 was consistent with the variation trend in anthocyanin content, suggesting that it might play an important role in anthocyanin accumulation in response to light exposure. Subcellular localization showed that PbrMATE9 was a membrane protein. More strikingly, the transient overexpression of PbrMATE9 promoted anthocyanin accumulation in the peel of pear, and the expression of structural genes (PbrCHI, PbrANS, PbrDFR, and PbrUFGT) in the anthocyanin biosynthesis pathway also increased significantly. Through co-expression network analysis, the transcription factors were identified, such as WRKY, COL, GATA, and BBX, which might be involved in the regulation of PbrMATE9. The study has enriched the genetic resources and improved the understanding of the regulation network of anthocyanin accumulation in pear.

Some cultivars of pear (Pyrus L.) show attractive red fruit skin due to anthocyanin accumulation. This pigmentation can be affected by environmental conditions, especially light. To explore the light-induced regulation network for anthocyanin biosynthesis and fruit coloration in pear, small RNA libraries and mRNA libraries from fruit skins of ‘Yunhongyihao’ pear were constructed to compare the difference between bagging and debagging treatments. Analysis of RNA-seq of fruit skins with limited light (bagged) and exposed to light (debagged), showed that PyPIF5 was down-regulated after bag removal. PymiR156a was also differentially expressed between bagged and debagged fruit skins. We found that PyPIF5 negatively regulated PymiR156a expression in bagged fruits by directly binding to the G-box motif in its promoter. In addition, PymiR156a overexpression promoted anthocyanin accumulation in both pear skin and apple calli. We confirmed that PymiR156a mediated the cleavage of PySPL9, and that the target PySPL9 protein could form heterodimers with two key anthocyanin regulators (PyMYB114/PyMYB10). We proposed a new module of PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10. When the bagged fruits were re-exposed to light, PyPIF5 was down-regulated and its inhibitory effect on PymiR156a was weakened, which leads to degradation of the target PySPL, thus eliminating the blocking effect of PySPL on the formation of the regulatory MYB complexes. Ultimately, this promotes anthocyanin biosynthesis in pear skin.

Gene & Accession Numbers: PyMYB10 (Pbr016663.1); PyMYB114 (Pbr042924.1); PyPIF5 (Pbr029607.1); PySPL9 (Pbr019232.1); PybHLH3 (Pbr017379.1); PyERF3 (Pbr030451.1).

Stone cells have been described to substantially influence pear fruit quality, as lignin and cellulose are the main components of stone cells. However, there are limited studies on the relationship between the variation and molecular basis of stone cells, lignin and cellulose content among different pear varieties. Here, to reveal the variation of stone cell content within different cultivated species, we collected 236 germplasms of sand pear (Pyrus pyrifolia) at 50 days after flower blooming (DAFB), the key stage of stone cell formation. In our results, we measured the content of stone cells, lignin and cellulose and found that these contents ranged from 2.82% to 29.00%, 8.84% to 55.30% and 11.52% to 30.55%, respectively. Further analysis showed that the variation coefficient of stone cell, lignin and cellulose content was 39.10%, 28.03% and 16.71%, respectively. Additionally, a significant correlation between stone cell, lignin and cellulose content were detected, and the correlation coefficient between the contents of stone cell and lignin (0.912) was higher than between the contents of stone cell and cellulose (0.796). Moreover, the average lignin content (29.73%) was higher than the average cellulose content (18.03%) in stone cells in pear fruits, indicating that lignin is the main component of stone cell in pears. Finally, on the basic of the transcriptome data, we identified 10 transcription factors belonging to bHLH, ERF, MYB, and NAC transcript families, which might be involved in lignin formation in stone cells. qRT-PCR experiments verified coincident trends between expression of candidate genes and stone cell content. This research laid foundation for future studies on genetic variation of stone cells in pear fruits and provided important gene resources for stone cell regulation.