Leaf senescence, an essential component of the plant life cycle, seriously affects the productivity of numerous commercial crops, with cytokinins serving as crucial regulators in delaying this process. Here, we observed that apple (Malus domestica) leaves exhibiting deficiencies in sorbitol synthesis due to antisense inhibition of ALOSE-6PHOSPHATE REDUCTASE (A6PR) presented an increase in cytokinin content and exhibited a delay in leaf senescence, in contrast to wild-type (WT) leaves. Transcriptome analysis indicated that the expression of cytokinin oxidase 7 (MdCKX7), encoding a key enzyme in the cytokinin degradation pathway, was significantly downregulated in the A6PR antisense lines. Functional verification confirmed that MdCKX7 facilitated the degradation of cytokinin and accelerated leaf senescence. Moreover, this leaf senescence phenotype was exacerbated by the co-expression of two DNA-binding One Zinc Finger (DOF) transcription factors, cycling DOF factor 3 (MdCDOF3) and MdDOF3.6, along with MdCKX7. Further biochemical and phenotypic analyses demonstrated that MdCDOF3 and MdDOF3.6 bind directly to the promoter region of MdCKX7, thereby transcriptionally activating its expression. Intriguingly, the expression of MdCDOF3, MdDOF3.6, and MdCKX7 is cooperatively induced by sorbitol. These findings demonstrate that the MdCDOF3/MdDOF3.6-MdCKX7 regulatory module orchestrates leaf senescence by facilitating cytokinin degradation in response to sorbitol signaling, revealing a mechanism by which sorbitol signaling modulates leaf senescence specifically through MdCKX7-mediated cytokinin degradation in apple plants.

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