School of Biological and Environmental Engineering, Chaohu University, Chaohu Regional Collaborative Technology Service Center for Rural Revitalization, Hefei, 23800, China; State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, China.
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, China.
Plant Physiol Biochem. 2024 Jun;211:108726. doi: 10.1016/j.plaphy.2024.108726. Epub 2024 May 11.
Tea is one of the most prevalent non-alcoholic beverages. The leaves of tea plants hyperaccumulate anthocyanins under cold stress, resulting in enhanced bitterness. Previously, we determined that the RING-type E3 ubiquitin ligase CsMIEL1 from the tea plant (Camellia sinensis (L.) O. Kuntze) is involved in the response to stress conditions. This study aimed to determine the role of CsMIEL1 in anthocyanin accumulation at the post-translational modification level. The results showed that the heterologous expression of CsMIEL1 led to an 86% decrease in anthocyanin levels, resulting in a significant decrease in the mRNA levels of related genes in Arabidopsis at low temperatures but no significant differences in other phenotypes. Furthermore, multi-omics analysis and yeast two-hybrid library screening were performed to identify potential downstream targets of CsMIEL1. The results showed that the overexpression of CsMIEL1 resulted in 45% (448) of proteins being differentially expressed, of which 8% (36) were downregulated in A.thaliana, and most of these differentially expressed proteins (DEPs) were clustered in the plant growth and secondary metabolic pathways. Among the 71 potential targets that may interact with CsMIEL1, CsMYB90 and CsGSTa, which are related to anthocyanin accumulation, were selected. In subsequent analyses, these two proteins were verified to interact with CsMIEL1 via yeast two-hybrid (Y2H) and pull-down analyses in vitro. In summary, we explored the potential mechanism by which the E3 ligase relieves anthocyanin hyperaccumulation at low temperatures in tea plants. These results provide a new perspective on the mechanisms of anthocyanin regulation and the molecular breeding of tea plants.
茶是最常见的无酒精饮料之一。茶树的叶子在寒冷胁迫下超积累花色素苷，导致苦味增强。此前，我们确定来自茶树（Camellia sinensis (L.) O. Kuntze）的 RING 型 E3 泛素连接酶 CsMIEL1 参与了对胁迫条件的反应。本研究旨在确定 CsMIEL1 在翻译后修饰水平上对花色素苷积累的作用。结果表明，CsMIEL1 的异源表达导致花色素苷水平降低 86%，导致低温下拟南芥相关基因的 mRNA 水平显著降低，但其他表型没有显著差异。此外，进行了多组学分析和酵母双杂交文库筛选，以鉴定 CsMIEL1 的潜在下游靶标。结果表明，CsMIEL1 的过表达导致 45%（448 个）的蛋白质表达差异，其中 8%（36 个）在拟南芥中下调，这些差异表达蛋白（DEPs）大多数聚类在植物生长和次生代谢途径中。在可能与 CsMIEL1 相互作用的 71 个潜在靶标中，选择了与花色素苷积累相关的 CsMYB90 和 CsGSTa。在随后的分析中，通过酵母双杂交（Y2H）和体外下拉分析验证了这两个蛋白与 CsMIEL1 相互作用。总之，我们探讨了 E3 连接酶在茶树低温下缓解花色素苷过度积累的潜在机制。这些结果为花色素苷调控机制和茶树的分子育种提供了新的视角。
