Silencing microRNA172 delays fruit ripening in apple (Malus domestica) through an AP2-ERF transcriptional regulation mechanism

MicroRNA172 (miR172) regulates many processes of plant development, including flowering and fruit growth, yet its function in fruit ripening remains unclear. In this study, we demonstrate that silencing miR172 in apple (Malus domestica) delays fruit ripening, which correlates with a postponed onset of climacteric ethylene production. At harvest, miR172-silenced fruit displayed lower respiration rates than wild-type (WT) fruit, although these rates re-aligned with WT after 12 weeks of cool storage. Although at harvest miR172-silenced and WT fruit showed similar firmness and levels of soluble solid content, the miR172-silenced fruit maintained greater firmness and a higher level of soluble solid content (SSC) after 12 weeks of post-harvest storage at 0oC. Comparative transcriptome analysis revealed these phenotypic changes to be associated with increased expression of APETALA2 (AP2) homologs MdAP2_1b/2a (miR172 target genes) and an Ethylene Response Factor (ERF) MdERF4, along with reduced expression of ethylene biosynthesis genes MdACS1/ACO1. In a dual luciferase reporter assay, MdAP2_1a/2a activated the MdERF4 promotor in tobacco leaves. These findings, together with existing knowledge that MdERF4 inhibits MdACS1/ACO1 expression, suggest a novel genetic regulatory network involving miR172, MdAP2 and MdERF4 in controlling apple fruit ripening.