Li Zhu, Han Yunyun, Li Xin, Zhao Jingjuan, Wang Nana, Wen Yangyang, Li Tongtong, Su Huangqiang, Gao Liping, Xia Tao, Liu Yajun
School of Life Science, Anhui Agricultural University, Hefei 230036, Anhui, China.
Lu'an Institute of Product Quality Supervision and Inspection, Lu'an City, China.
Hortic Res. 2024 May 5;11(7):uhae136. doi: 10.1093/hr/uhae136. eCollection 2024 Jul.
Flavonoids constitute the main nutraceuticals in the leaves of tea plants (). 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 encoding a mitogen-activated protein kinase in leaves. Luciferase complementation and yeast two-hybrid assays disclosed that CsMPK4a interacted with CsWD40. Phosphorylation assay 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 and phosphorylated CsWD40 variants, which were introduced into the Arabidopsis mutant. Metabolic analysis showed that the anthocyanin and proanthocyanidin content was lower in transgenic plants than transgenic and wild type plants. The transient overexpression of downregulated the anthocyanidin biosynthesis in tea leaves. The dual-fluorescein protein complementation experiment showed that CsWD40 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.
黄酮类化合物是茶树叶片中的主要营养成分。迄今为止，尽管已知干旱胁迫会对茶叶中黄酮类化合物的生物合成产生负面影响，但这一现象背后的机制尚不清楚。在此，我们报道了一种蛋白质磷酸化机制，该机制在干旱条件下对茶叶中黄酮类化合物的生物合成起负调控作用。转录分析显示，叶片中黄酮类化合物生物合成的基因表达下调，而编码丝裂原活化蛋白激酶的基因表达上调。荧光素酶互补和酵母双杂交试验表明，CsMPK4a与CsWD40相互作用。磷酸化试验、特异性蛋白质免疫和蛋白质质谱分析表明，CsWD40的Ser-216、Thr-221和Ser-253是CsMPK4a的潜在磷酸化位点。此外，蛋白质免疫分析发现干旱条件下茶叶中CsWD40的磷酸化水平增加。这三个磷酸化位点的突变产生了去磷酸化的CsWD40和磷酸化的CsWD40变体，并将其导入拟南芥突变体中。代谢分析表明，转基因植物中的花青素和原花青素含量低于转基因植物和野生型植物。CsWD40的瞬时过表达下调了茶叶中花青素的生物合成。双荧光素蛋白互补实验表明，CsWD40不与CsMYB5a和CsAN2相互作用，这两个是茶树中原花青素和花青素生物合成的关键转录因子。这些发现表明，CsMPK4a对CsWD40的磷酸化在干旱胁迫下下调了茶树中黄酮类化合物的生物合成。
