The phosphorylation of a WD40-repeat protein negatively regulates flavonoid biosynthesis in Camellia sinensis under drought stress

Abstract

Flavonoids constitute the main nutraceuticals in the leaves of tea plants (Camellia sinensis). To date, although it is known that drought stress can negatively impact the biosynthesis of flavonoids in tea leaves, the mechanism behind this phenomenon is unclear. Herein, we report a protein phosphorylation mechanism that negatively regulates the biosynthesis of flavonoids in tea leaves in drought conditions. Transcriptional analysis revealed the downregulation of gene expression of flavonoid biosynthesis and the upregulation of CsMPK4a encoding a mitogen-activated protein kinase in leaves. Luciferase complementation and yeast two-hybrid assays disclosed that CsMPK4a interacted with CsWD40. Phosphorylation assay in vitro, specific protein immunity, and analysis of protein mass spectrometry indicated that Ser-216, Thr-221, and Ser-253 of CsWD40 were potential phosphorylation sites of CsMPK4a. Besides, the protein immunity analysis uncovered an increased phosphorylation level of CsWD40 in tea leaves under drought conditions. Mutation of the three phosphorylation sites generated dephosphorylated CsWD40 3A and phosphorylated CsWD40 3D variants, which were introduced into the Arabidopsis ttg1 mutant. Metabolic analysis showed that the anthocyanin and proanthocyanidin content was lower in ttg1:CsWD40 3D transgenic plants than ttg1::CsWD40 3A transgenic and wild type plants. The transient overexpression of CsWD40 3D downregulated the anthocyanidin biosynthesis in tea leaves. The dual-fluorescein protein complementation experiment showed that CsWD40 3D did not interact with CsMYB5a and CsAN2, two key transcription factors of procyanidins and anthocyanidins biosynthesis in tea plant. These findings indicate that the phosphorylation of CsWD40 by CsMPK4a downregulates the flavonoid biosynthesis in tea plants in drought stresses.

Acknowledgements

This work was financially supported by Anhui Provincial Natural Science Foundation (2308085MC94, 202204c06020035), the joint funds of National Natural Science Foundation of China (U21A20232), and the Natural Science Foundation of China (32000366, 32372756), the National Key Research and Development Program of China (2022YFF1003103).

Data availability

All data generated from the study appear in the submitted article.

Conflict of interest statement

The authors declare no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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