ATAC-seq and RNA-seq reveal the role of AGL18 in regulating fruit ripening via ethylene-auxin crosstalk in papaya

Papaya (Carica papaya) is the third most-produced tropical fruit. It is a climacteric fruit, which is extremely perishable due to rapid physiological disintegration. The compound 1-methylcyclopropene (1-MCP) is an efficient ethylene receptor inhibitor that can interact with ethylene receptors and avoid its physiological activation. In this study, ATAC-seq was exploited to detect the accessible open chromatin of fruit after different 1-MCP treatments. The results revealed that the differential peaks of accessible open chromatin were enriched in the intergenic and promoter regions. MADS and AP2/ERF-ERF accounted for a large proportion among the various differential regulated transcription factors identified. KEGG enrichment analysis showed that plant hormone signaling is critical in papaya ripening under 1-MCP treatment. Integrated analysis of the ATAC-seq and RNA-seq data was performed to establish the expression profiles of ripening related genes under short-term and long-term 1-MCP treatments. Multiple transcriptional factors, including MADS (CpAGL18), and eight ethylene and auxin signal pathway-related differentially expressed genes (DEGs) were found associated with fruit softening and ripening. Interestingly, CpAGL18 expression significantly increased during ripening under control condition but was strongly suppressed under long-term 1-MCP treatment. The promoter analysis predicted cis-acting elements of MADS in CpACS1 and CpSAUR32. In addition, EMSA (Electrophoretic mobility shift assay), Y1H (Yeast one hybrid) and ChIP-qPCR (Chromatin immunoprecipitation and quantitative PCR) assays verified CpAGL18 binding to the promoters of CpACS1 and CpSAUR32 in vivo and in vitro. Finally, the dual-luciferase reporter assay revealed that CpAGL18 activated the promoters of CpACS1 and CpSAUR32. These findings suggest that CpAGL18 plays a key role in fruit softening and ripening in papaya by integrating ethylene and auxin signaling.