iCUT&Tag-based pan-fruit epigenomic profiling identifies recurrent H3K9 acetylation enrichment during fleshy fruit ripening

Fleshy fruit ripening involves broadly convergent physiological changes across phylogenetically distinct species, yet the extent to which shared chromatin-level mechanisms underlie this convergence remains poorly understood, partly due to technical challenges posed by metabolite-rich fruit tissues. Here, we develop iCUT&Tag, an improved chromatin profiling method requiring as few as 500 nuclei, and apply it to generate histone modification profiles across 12 fruit species spanning 9 botanical families, demonstrating its broad application in metabolite-rich fruit tissues. Comparative profiling of climacteric (tomato, apple) and non-climacteric (citrus, strawberry) fruit identifies H3K9 acetylation (H3K9ac) as a recurrently enriched chromatin feature associated with ripening-linked gene activation, regardless of the dominant hormonal system. In tomato, SlHDA1 loss is accompanied by premature H3K9ac accumulation at ripening-associated loci, whereas in strawberry, FaHDA19 silencing is associated with increased H3K9ac at transcriptionally activated ripening-related loci. Mechanistically, comparative genetic analyses suggest a bipartite regulatory architecture: H3K9ac-mediated permissive chromatin state appears conserved across lineages, yet productive transcription additionally requires species-specific transcription factor activity-SlNOR in tomato and FaMYB10 in strawberry-to drive phenotypically convergent ripening. Our study provides a validated technological framework for plant epigenomics and identifies a recurrent associated chromatin-level feature that may underlie the convergent evolution of fleshy fruit ripening.