The miR166 targets CsHDZ3 genes to negatively regulate drought tolerance in tea plant (Camellia sinensis).

Drought is the stressor with a significant adverse impact on the yield stability of tea plants. HD-ZIP III transcription factors (TFs) play important regulatory roles in plant growth, development, and stress responses. However, whether and how HD-ZIP III TFs are involved in drought response and tolerance in tea plants remains unclear. Here, we identified seven HD-ZIP III genes (CsHDZ3-1 to CsHDZ3-7) in tea plant genome. The evolutionary analysis demonstrated that CsHDZ3 members were subjected to purify selection. Subcellular localization analysis revealed that all seven CsHDZ3s located in the nucleus. Yeast self-activation and dual-luciferase reporter assays demonstrated that CsHDZ3-1 to CsHDZ3-4 have trans-activation ability whereas CsHDZ3-5 to CsHDZ3-7 served as transcriptional inhibitors. The qRT-PCR assay showed that all seven CsHDZ3 genes could respond to simulated natural drought stress and polyethylene glycol treatment. Further assays verified that all CsHDZ3 genes can be cleaved by csn-miR166. Overexpression of csn-miR166 inhibited the expression of seven CsHDZ3 genes and weakened drought tolerance of tea leaves. In contrast, suppression of csn-miR166 promoted the expression of seven CsHDZ3 genes and enhanced drought tolerance of tea leaves. These findings established the foundation for further understanding the mechanism of CsHDZ3-miR166 modules' participation in drought responses and tolerance.