CpMYB114-like enhances CpNADP-ME2 expression to induce malate reduction and sugar accumulation during papaya ripening

Sugars and organic acids form the core fundamental flavor foundation of ripe papaya fruit. Although R2R3-type v-myb myeloblastosis viral oncogene homolog transcription factors (R2R3-MYB TFs) have been reported to play important roles in regulating the primary and secondary metabolism in fruits. However, the functions and underlying regulatory mechanisms of R2R3-MYB TFs in the metabolism of sugars and organic acids during papaya ripening remain unclear. In this study, through RNA sequencing (RNA-seq) coupled with quantitative real-time polymerase chain reaction (qRT-PCR) validation, a novel ethylene-responsive R2R3-MYB TF gene, CpMYB114-like ( CpMYB114L ), was identified. Transcriptome analysis and ethylene induction assays highlighted CpMYB114L as the key MYB gene activated during papaya ripening. Subcellular localization experiments confirmed that CpMYB114L was localized in the nucleus. Analyses of transgenic papaya fruits and calli, demonstrated that CpMYB114L promoted the accumulation of glucose, fructose, and sucrose, while simultaneously accelerating malate degradation. DNA Affinity Purification sequencing (DAP-seq) combined with yeast one-hybrid (Y1H) assays identified that CpMYB114L directly bound to the promoters of two sugar/organic acid metabolism-related genes, Isocitrate dehydrogenase 5 ( CpIDH5 ) and NADP-dependent malic enzyme 2 ( CpNADP-ME2 ). Electrophoretic mobility shift (EMSA), dual-luciferase reporter (Dual-LUC) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays further confirmed that CpMYB114L positively activated the expression of CpIDH5 and CpNADP-ME2 by directly binding to the specific regions of their promoters. Notably, transient overexpression of CpNADP-ME2 in papaya fruits and calli recapitulated decreased malate levels and increased sugar content, whereas CpIDH5 overexpression did not exhibit any functional relevance. This study elucidates a regulatory module, CpMYB114L- CpNADP-ME2 , linking malate catabolism and sugar accumulation, offering valuable insights into flavor improvement strategies for papaya.