Genome-Wide Identification of CmPOD Genes and Partial Functional Characterization of CmPOD52 in Lignin-Related Granulation of ‘Sanhong’ Pomelo (Citrus maxima)

The granulation of pomelo (Citrus maxima) juice sacs severely compromises fruit quality and is closely associated with lignin accumulation, a process catalyzed by peroxidases (PODs). Analysis of ‘Sanhong’ pomelo juice sacs collected 175–215 days after flowering revealed that bound peroxidase (BPOD) activity paralleled changes in lignin content, suggesting a potential role for BPOD in lignin biosynthesis. A total of 71 CmPOD genes were identified in the pomelo genome through integrated HMMER and BLAST analyses. Among them, CmPOD52 was selected for functional characterization based on its alkaline peroxidase properties, absence of a CE domain, predicted extracellular localization, and gradually increasing expression pattern revealed by RT-qPCR. Its transient overexpression in ‘Sanhong’ pomelo juice sacs for 36 h increased BPOD activity 2.06-fold (p < 0.01) compared to the empty vector control, indicating that CmPOD52 may be a BPOD gene. The recombinant CmPOD52 protein was expressed in a prokaryotic system, purified, and used in enzymatic assays with sinapyl alcohol as the substrate. The recombinant CmPOD52 protein, assayed at 272 nm with controls (substrate-only blank and heat-inactivated protein), showed an activity of 13.67 ± 0.9 U. The experimental group showed new products, identified by mass spectrometry as sinapyl alcohol dimers, thus suggesting that the recombinant protein catalyzes the dehydrogenation and polymerization of sinapyl alcohol monomers. This study identified CmPOD52, a gene potentially involved in lignin polymerization in pomelo juice sacs, offering a key candidate for further in vivo validation.