MdMAPK6-mediated phosphorylation of MdWRKY9 regulates apple fruit ripening through interaction with MdERF5L

Abstract

WRKY transcription factors are essential for mediating many developmental processes, such as fruit ripening, a highly controlled and intricate physiological phenomenon. In the current research, a novel transcription factor, MdWRKY9, was identified and categorized, which significantly promotes apple fruit ripening. Its role was validated through a combination of transient injection and stable overexpression-transformed tomato experiments. Notably, MdWRKY9 interacts with the fruit ripening suppressor MdERF5L at protein and DNA levels. This interaction counteracts MdERF5L-mediated MdACS1 repression. MdACS1 is an important ethylene biosynthesis enzyme; the mentioned process eventually allows fruit ripening. Furthermore, phosphorylation, a post-translational modification, regulates maturation and ethylene synthesis. Through liquid chromatography-tandem mass spectrometry, we identified phosphorylation sites within the MdWRKY9-GFP protein. MdMAPK6-MdWRKY9 interaction and MdMAPK6-mediated phosphorylation of MdWRKY9 were confirmed through protein-protein interaction assays, such as bimolecular fluorescence complementation (BIFC), yeast two-hybrid (Y2H), protein phosphorylation, luciferase complementation imaging (LCI), and pull-down assays. Specifically, MdMAPK6 phosphorylates MdWRKY9 at Tyr394 site, enhancing the stability and activity of MdWRKY9 and thus modulating its regulatory role in fruit maturation. These findings provide new directions and insights into the intricate regulatory network governing the ripening of apples.

Acknowledgments

The National Natural Science Foundation of China (Grant No. 32172533), Agricultural Variety Improvement Project of Shandong Province (Grant Nos 2021LZGC024, 2022LZGC010), Earmarked Fund for Modern Agro-industry Technology Research System, China (CARS-27), National Key Research and Development Program of China (2023YFD2301000), Postdoctoral Fellowship Program of CPSF (GZC20232159), and General Program of China Postdoctoral Science Foundation (2024M762645) provided funding for our research.

Author contributions

N.W., X.-S.C., H.W., and S.-H.Z. came up with the idea for the study. The experiments were carried out by S.-H.Z., H.W., Y.-C.F., J.-H.Z., T.W., and W.-J.L., who also supplied the materials and reagents and conducted the data analysis. The paper was prepared by S.-H.Z. and H.W.

Data availability

All supporting data are included in this paper or the supplementary materials.

Conflict of interest statement

The writers of this paper have no conflicts of interest that they must disclose.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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