Two DNA-binding One Zinc Finger transcription factors, MdCDOF3 and MdDOF3.6, accelerate leaf senescence by activating cytokinin oxidase MdCKX7 in response to sorbitol signaling in apple

Abstract

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.

Acknowledgements

We would like to express our sincere gratitude to Professor Lailiang Cheng at Cornell University for generously providing the A6PR silencing transgenic apple materials. This work was supported by grants from the National Key Research and Development Program of China (2023YFD2301000, 2022YFD2100100), the National Natural Science Foundation of China (32122080), the Key Research and Development Program of Shandong Province (2023CXGC010709), the Taishan Scholar Project Special Funds of China (Grant No.tsqnz20231206), and the Natural Science Foundation of Shandong Province (ZR2024QC255).

Author contributions

D.G.H. and T.T.Z. conceived and designed the study. W.J.Z. performed the experiments, W.J.Z., C.N.M., Y.X., F.X, Y.T.L., C.K.W., and W.K.L. analyzed the data. D.G.H., T.T.Z., and W.J.Z. wrote the paper. All authors discussed the results and commented on the manuscript.

Data availability

All data generated or analyzed during this study are provided in the article and its supplementary data files.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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