Plants undergo various age-dependent changes in leaf morphology during juvenile to adult vegetative stage. However, the precise molecular mechanisms governing these changes in apple (Malus domestica) remain unknown. Here, we showed that CYTOKININ OXIDASE/DEHYDROGENASE5 (MdCKX5), an age-dependent gene, encodes a functional CKX enzyme and serves as the common downstream target of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MdSPL14 and WRKY transcription factor MdWRKY24 to control the degradation of cytokinin (CK).

 

As the target of mdm-microRNA156a, MdSPL14 interacts with MdWRKY24 to coordinately repress the transcription of MdCKX5 by forming the age-mediated mdm-miR156a–MdSPL14–MdWRKY24 module, which regulates age-dependent changes in CK during the juvenile-to-adult phase transition. We further demonstrated that MdARR6, a type-A ARABIDOPSIS RESPONSE REGULATOR (ARR), is a negative feedback regulator in the CK signaling pathway. Silencing of MdARR6 in apple resulted in large leaves with smaller epidermal cells and a greater number of epidermal cells.

 

Biochemical analysis showed that the mdm-miR156a–MdSPL14–MdWRKY24 module acts as a transcriptional repressor to directly regulate MdARR6 expression, thus controlling the age-dependent changes in leaf size by reducing CK responses. These findings established a link between the age pathway and CK signaling and revealed the molecular mechanism underlying age-dependent changes during the juvenile-to-adult phase transition; our results also provide targets for the genetic improvement of the vegetative phase transition in apple.