Genome-wide identification of the PPO gene family in pear and transcriptional analysis of its members involved in fruit flesh browning

【Objective】Pear (Pyrus L.) is an economically important fruit crop worldwide, with significant nutritional and health- promoting properties due to its abundant phenolic compounds. However, postharvest fruit browning, particularly in fresh-cut products, severely impairs commercial quality and shelf life, representing a major constraint for the pear industry. Enzymatic browning, which is primarily mediated by polyphenol oxidase (PPO), occurs when cellular compartmentalization is disrupted upon tissue damage, allowing PPO to oxidize endogenous phenolic substrates to highly reactive quinones that subsequently polymerize into dark pigments. Despite the pivotal role of PPO in fruit browning, the PPO gene family in pear has not been systematically characterized across multiple representative germplasms, and the evolutionary dynamics, structural diversity, and functional differentiation of its mem-bers remain poorly understood. This study aims to characterize the PPO gene family in four representative pear varieties, including Yunhong No.1 (sand pear), Dangshansu (white pear), Max Red Bartlett (European pear) and Shanxiduli (wild pear), to analyze their physicochemical properties, and to explore the expression patterns of PPO members during fruit development and in different tissues.【Methods】 High-quality genome assemblies of the four pear species were obtained from the GDR database and the Pear Genome Database. PPO candidate genes were initially screened using HMMER 3.0 with the PPO characteristic domain (PF00264) from the Pfam database, followed by BLAST homology searches (Evalue ＜ 10-5 ). All candidate sequences were further validated using NCBI-CDD and Pfam tools to confirm the presence of conserved PPO domains. Physicochemical properties, including polypeptide length, molecular weight, theoretical isoelectric point (pI), instability index, and grand average of hydropathicity (GRAVY), were predicted using ExPASy. Subcellular localization was determined via Plant-mPLoc, and three-dimensional protein structures were modeled using Swiss-Model. Chromosomal localization and interspecific synteny analysis were performed using TBtools, while phylogenetic trees were constructed using MEGA 11 with the maximum-likelihood (ML) method and 1000 bootstrap replicates. Gene structures were visualized using GSDS 2.0, and conserved motifs were identified using MEME. Promoter regions (2 kb upstream of the start codon) were analyzed via PlantCARE to identify cis-acting regulatory elements. Transcriptome data from fresh-cut pear fruits of the browning-resistant cultivar Hongxiangsu (HXS) and the susceptible cultivar Mantianhong (MTH) were generated by RNA-seq and analyzed to map the expression of genes involved in phenylpropanoid and tyrosine metabolic pathways. Additionally, RT-qPCR was performed to validate the expression dynamics of the core gene PpyPPO10 at different fruit developmental stages (70-120 days after full bloom) and in various tissues including pedicel, receptacle, anther, sepal, petiole, leaf, peel, and flesh. All RT-qPCR experiments included three biological replicates and three technical replicates, with PpyTubulin as the internal reference gene, and relative expression levels were calculated using the 2-ΔΔCt method.【Results】A total of 8-10 conserved PPO genes were identified, predominantly clustered at the terminal regions of Chr05 and Chr10. The encoded proteins were hydrophilic, with 586- 941 amino acids, varying molecular weights, isoelectric points (5.39-8.40), and instability indices (37.85-52.21); most were predicted to localize to the cytoplasm, chloroplasts, or nucleus, except one extracellular member. Phylogenetic analysis divided all pear PPOs into nine subgroups, with the majority falling into Group I. Pear PPO genes shared high homology with those of apple and peach, but diverged greatly from non-Rosaceae crops. Strong syntenic blocks were detected between pear Chr05/Chr10 and apple Md5/Md10, whereas collinear fragments with peach and strawberry were scattered due to differing chromosome numbers. Ten conserved motifs and canonical PPO catalytic domains were identified, and gene promoters harbored abundant cis-elements responsive to light, phytohormones, and abiotic stresses. Combined with transcriptome data from the sand pear Yunhong No.1, it was confirmed that pear PPOs modulate the biosynthesis of caffeic acid, chlorogenic acid, ferulic acid, and other phenolics through the tyrosine and phenylpropane pathways. The core gene PpyPPO10 showed the closest evolutionary relationship with apple MdPPO, and also clustered closely with PPO homologs from other woody plants, including peach, poplar, and tea. RT-qPCR results revealed that PpyPPO10 expression was significantly higher in the browningresistant cultivar Hongxiangsu than in the sensitive Mantianhong, with maximal transcript abundance in flesh and moderate levels in peel.【Conclusion】This study systematically characterizes the PPO gene family in four representative pear species, revealing that 8-10 conserved members, which share high evolutionary conservation with Rosaceae homologs, are organized as tandem arrays on Chr05 and Chr10, and encode hydrophilic proteins with typical PPO domains. The core gene PpyPPO10, identified as a key candidate regulating flesh browning, integrates signals from light, phytohormones, and abiotic stresses through its promoter, and coordinately modulates phenolic biosynthesis via the tyrosine and phenylpropanoid pathways. Its enhanced expression in browning-resistant cultivars and flesh-specific accumulation pattern underscore its pivotal role in browning regulation. These findings provide essential candidate genes and theoretical support for molecular breeding aimed at developing browning-resistant pear varieties, and lay a foundation for further functional validation of PPO genes in fruit quality regulation.