Differential Accumulation of Apple Scar Skin Viroid Drives Uneven Apple Coloration via Disruption of the Defense–Flavonoid Balance

Apple scar skin viroid (ASSVd) induces uneven fruit coloration, which severely compromises commercial value. However, the molecular mechanisms underlying this viroid-induced pigmentation disorder remain unclear. To address this, we performed an integrated analysis combining physiology, biochemistry, cytology, and multi-omics approaches. The results showed that ASSVd infection severely suppressed anthocyanin accumulation in apple peel, particularly in the nonpigmented sectors (Vd-G, approximately 6.95 nmol/g), which exhibited levels dramatically lower than those in the pigmented sectors of infected peel (Vd-R, approximately 221.04 nmol/g) and the ASSVd-uninfected control (CK, approximately 317.73 nmol/g). In contrast, chlorophyll content was significantly higher in ASSVd-infected tissues compared with the control, with no notable difference between Vd-G and Vd-R sectors. Ultrastructural analysis by transmission electron microscopy showed a lack of chromoplasts in infected tissues, revealing that ASSVd interferes with the normal chloroplast-to-chromoplast transition, a process essential for pigment development. Notably, Vd-G sectors exhibited higher viroid titers, along with elevated activities of polyphenol oxidase and peroxidase. Multi-omics analysis indicated titer-dependent reprogramming of flavonoid biosynthesis, involving altered expression of key genes (e.g., MdCHS and MdF3H) and metabolites (e.g., cyanidin 3-arabinoside cation and cyanidin-3-O-glucoside), collectively suppressing anthocyanin accumulation. Furthermore, the WRKY transcription factor family was specifically enriched in the high-titer Vd-G tissues relative to Vd-R, suggesting its regulatory role in these titer-dependent metabolic shifts. Joint pathway analysis additionally identified co-enrichment in pathways related to linoleic acid metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis, and cutin, suberine, and wax biosynthesis specifically in Vd-G. In summary, this study reveals a multilevel regulatory network in which local viroid titer drives coloration defects by disrupting the balance between host defense and flavonoid metabolism. The specific enrichment of WRKY transcription factors provides an important transcriptional clue for understanding this titer-dependent metabolic reprogramming.