CsMIEL1 effectively inhibits the accumulation of anthocyanins under low temperatures in tea plants (Camelliasinensis).

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.