Bitter pit disorder development: Evidence from transcriptomic and cellular analysis in different tissues

Bitter pit (BP), a physiological disorder, causes significant losses to the apple industry. The precise mechanisms underlying BP remain incompletely understood. Herein, transcriptomic analysis was carried out on healthy flesh (FH), flesh between healthy and disordered (FB), disordered flesh (FD), healthy pericarp (PH), and disordered pericarp (PD). Results demonstrated that differentially expressed genes (DEGs) are involved in programmed cell death (PCD), calcium signaling and transport, cell wall degradation, respiration, and redox. The signal transduction pathways of gibberellic acid, jasmonic acid, salicylic acid, abscisic acid, cytokinin, auxin, brassinosteroid, and ethylene were also implicated. Among these, DEGs enriched in auxin signaling were particularly up-regulated in FD. WRKY, MYB, bZIP, and NAC transcription factors may play significant regulatory roles in the BP formation. The different fold changes of DEGs in FD vs FB comparison and FD vs FH implied the coordination of local and long-distance responses during BP development. Results from fluorescence staining, transmission electron microscopy and DNA ladder indicated that PCD may occur during BP development. PCD-related enzyme (responsive to desiccation-21) and genes (MdCEPI-1 and MdCEPI-2) were higher in FD and PD, whereas MdDAD1-1 and MdDAD-2 were significantly down-regulated in FD. Genes encoding cytochrome P450, squalene cyclase, and glutathione S-transferase, respectively, emerged as potential candidates linked to BP development, given their known roles in bitter compound biosynthesis and stress-related cell death pathways. Moreover, in both pericarp and flesh tissues, disordered samples exhibited increased accumulation of reactive oxygen species.