Integrated Metabolomics and Targeted Gene Expression Profiling Reveal the Arginine–Anthocyanin Axis in Pomegranate Aril Paleness Disorder

Aril paleness (AP) is a new physiological disorder of pomegranate (<i>Punica granatum</i> L.) characterized by pale, dry and tasteless arils, while the peel remains healthy-looking. Its molecular basis is unknown. We used an integrated metabolomic and targeted gene expression approach on arils from four Iranian cultivars displaying no to severe AP symptoms. LC-MS profiling detected 617 reliable metabolites, with 266 metabolites consistently reduced in all symptomatic samples. Enrichment analysis revealed that arginine biosynthesis, glutathione metabolism and primary amino acid metabolism were the processes most strongly affected by AP. Protein interaction network analysis indicated that the arginine degradation pathway is the primary down-regulated module that interacts with the anthocyanin biosynthetic machinery, primarily through phenylalanine ammonia-lyase (PAL) hubs. Based on this network, seven genes representing both pathways were selected for targeted expression analysis. The qPCR analysis showed strong repression of arginase (<i>PgADS</i>, XM-031537872), aldehyde dehydrogenase (<i>PgAL12A1</i>, XM-031551051) and anthocyanin synthase (<i>PgOXKF</i>, KF841619.1) in the cultivar 'Torud' exhibiting severe AP symptoms compared with the symptom-free cultivar 'Damavand'. In contrast, phenylalanine ammonia-lyase (<i>PgPAL1</i>, KY094504.2) was unexpectedly induced 33-fold in the cultivar 'Torud', while the downstream anthocyanin-related UDP-glucosyltransferase (<i>PgUGT</i>, MK058491.1) remained unchanged. These findings suggest that the collapse of arginine metabolism, combined with the downstream blockage of anthocyanin biosynthesis, underlies AP. These findings provide the first molecular insights into the mechanisms underlying AP, offering a basis for breeding and post-harvest strategies aimed at enhancing pomegranate's AP tolerance.