Browning of olive (Olea europaea L.) fruit reduces the sensory and nutritional qualities of olive oil, thereby increasing production costs. Polyphenol oxidases (PPOs) are the key enzymes that catalyze phenolic substance oxidation and mediate enzymatic browning in olive fruit, but the exact regulatory mechanism remains unclear. The main challenge is the lack of comprehensive information on OePPOs at the genome-wide level. In this study, 18 OePPO genes were identified. Subsequently, we performed a bioinformatic analysis on them. We also analyzed the expression patterns and determined the relationship among browning degree, PPO activity, and expression of OePPOs in the fruits of three olive varieties. Based on our analysis, we identified the four most conserved motifs. OePPOs were classified into two groups, with OePPOs from Group 1 showing only diphenolase activity and OePPOs from Group 2 exhibiting both mono-/diphenolase activities. Seven pairs of gene duplication events were identified, and purifying selection was found to have played a critical role in the evolution of the OePPO gene family. A positive correlation was observed between the browning degree of olive fruit and PPO activity across different olive varieties. Moreover, two important genes were found: OePPO-5 the main effector gene responsible for fruit browning, and OePPO-8, a key gene associated with specialized metabolite synthesis in the olive fruit. In short, our discoveries provide a basis for additional functional studies on OePPO genes and can help elucidate the mechanism of enzymatic browning in olive fruit in the future.

Hydroxytyrosol is a natural phenolic compound found in olives. Phenylacetaldehyde reductase (PAR) is a key enzyme in the final step of the hydroxytyrosol biosynthesis pathway in olives. However, genome-wide studies on the PAR gene family in olives have not been reported. In this study, 21 genes were identified through a genome-wide analysis. Phylogenetic analysis classified these genes into three subgroups: PAR, CCR (Cinnamoyl-CoA reductase), and DFR (Dihydroflavonol 4-reductase). Expression pattern analysis suggested that genes within these subfamilies may play crucial roles in the biosynthesis of polyphenols, lignin, and anthocyanins, respectively. Three-dimensional structural modeling and molecular docking of the OePAR1 revealed that hydrogen bonds, hydrophobic interactions, and π–π stacking interactions collectively influence the affinity between PAR and its substrates. Residues at the active site form hydrogen bonds, with variations contributing to substrate specificity. The substrate with the strongest affinity for OePAR1 was identified as 3,4-dihydroxyphenylacetaldehyde (3, 4-DHPAA), with a binding energy of −4.98 kcal/mol, in agreement with previous enzymatic activity validation. Subcellular localization studies revealed that OePAR1 is localized to the chloroplast. This study provides essential insights into the biological functions of OePARs in olives and lays the groundwork for enhancing olive oil quality through genetic engineering.