The miR156k-SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE2 module regulates the abscisic acid receptor PYL2 during citrus fruit ripening

Abstract Members of the microRNA156 (miR156) family play essential roles in various biological processes, yet their involvement in regulating fruit ripening in citrus remains poorly understood. In this study, we determined that the miR156k- SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 2 (SPL2) module plays an important role in regulating citrus fruit ripening. Heterologous expression of csi-miR156k in Mini-Citrus Hongkong kumquat (Fortunella hindsii) significantly promoted fruit ripening, with the same phenotype observed when SPL2 was transiently silenced. Phenotypic analysis of F. hindsii transgenic lines revealed that SPL2 is the key target gene through which miR156k regulates fruit ripening in citrus. We used DNA affinity purification sequencing (DAP-Seq) and interaction assays to identify PYRABACTIN RESISTANCE LIKE 2 (PYL2) as the downstream target gene of SPL2, showing that PYL2 is negatively regulated by SPL2. Transient expression or repression of PYL2 in F. hindsii fruit greatly altered the duration of fruit color break. Moreover, screening of a yeast two-hybrid library revealed that caffeoyl CoA methyltransferase 7 (CCoAOMT7) directly interacts with SPL2. CCoAOMT7 recruits S-adenosyl-L-homocysteine hydrolase1 (SAHH1), leading to alterations in the DNA and histone methylation levels of the PYL2 promoter. Overall, this study reveals a previously uncharacterized mechanism by which the miR156k-SPL2 module regulates citrus fruit ripening by modulating PYL2 through epigenetic regulation.