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.

Figure 1

Effects of drought stress on the biosynthesis of flavonoid in tea leaves. A Diagram of flavonoid biosynthetic pathway. B Expression level of structural and key transcription regulatory genes in flavonoid pathway of tea plant under drought stress by 20% PEG4000 for 24 h, 48 h, 72 h. The FPKM values of gene expression at different time points are listed in Data S3 (see online supplementary material). C The expression levels of key genes in the flavonol and catechin biosynthesis pathways in tea leaves were verified by qRT-PCR under drought stress at 48 h. The gene expression level data from 0–48 hours are listed in Fig. S2 (see online supplementary material). D The catechin content in tea leaves under drought stress at 48 h. The relative variation in phenolic compound content during the 0–48 h drought treatment is shown in Data SS1.

Figure 2

Interaction between CsMPK4a and proteins regulating the flavonoid biosynthesis by Yeast two-hybrid and dual luciferase assay. A Screening of CsMPK4a interacting proteins involved in regulation of flavonoid biosynthesis in tea plants. B A dual luciferase assay in N. benthamiana leaves shows that CsWD40 interacts with CsMPK4a. MPK4a-C represents CsMPK4a-cLUC; WD40-N, CsWD40-nLUC; C, cLUC empty; N, nLUC empty. –WL, yeast growth on medium without Trp and Leu. –WLHAde, yeast growth on medium lacking Trp, Leu, His, and Adenine.

Figure 3

Identification of CsMPK4a-mediated phosphorylation sites on CsWD40 in vitro.  A Prokaryotic expression proteins of CA-CsMPK4a and verification by immune blot with His and MBP antibodies. B LC–MS/MS spectrum of the CsWD40 peptide containing serine 216, threonine 221, serine 253 phosphorylated by CsMPK4a. C Schematic diagram of the sites on CsWD40 protein potentially phosphorylated by CsMPK4a. D Phosphorylation reaction between CA-CsMPK4a and CsWD40 in vitro, and immune blot with anti-P-S216, anti-P-T221 and anti-P-S253 bodies. E CsWD40 and CsMPK4a in the enzyme reaction solution were identified by Coomassie blue staining.

Figure 4

Phenotype and proanthocyanidin content analysis of tri-site phosphorylated and dephosphorylated CsWD40 transgenic Arabidopsis thaliana.  A Phenotype of ttg1::CsWD40^3A and ttg1::CsWD40^3D transgenic A. thaliana plants at seedling stage. B Phenotype of the transgenic A. thaliana at the rosette stage. C Proanthocyanidin content in green fruit pod of the transgenic plants. D The color of seed coat and proanthocyanidin accumulation of transgenic mature seeds. The accumulation of proanthocyanidins in the seeds can be visualized by staining with DMACA.

Figure 5

The effect of CsWD40 phosphorylation on the interaction between CsWD40 and CsMYB5a, CsAN2 was assessed through BiFC assay. A and B The interaction between CsWD40, CsWD40^3A, and CsWD40^3D with CsMYB5a (A) and CsAN2 (B) was examined by BiFC assay in Arabidopsis thaliana protoplasts. P in the figure is the positive control group.

Figure 6

The dephosphorylation of CsWD40 negatively regulates anthocyanin biosynthesis in tea plants under stress. A and D Transgene verification detected by qRT-PCR. B Effect of transient overexpression of CsWD40^3D on anthocyanin biosynthesis in tea leaves. C The relative area of pink ring in control group and treatment group of transient overexpression of CsWD40^3D. E Effect of transient overexpression of CsWD40^3A on anthocyanin biosynthesis in tea leaves. F The relative area of pink ring in control group and treatment group of transient overexpression of CsWD40^3A. The anthracnose infection-induced anthocyanin biosynthesis system was employed to examine the effect of overexpressing the CsWD40^3D and CsWD40^3A gene on anthocyanin biosynthesis in tea leaves.