Mechanical harvesting in the tea industry has become increasingly essential due to its advantages in increasing productivity and reducing labor costs. Leaf droopiness caused a high rate of broken leaves, hindering the mechanized harvesting quality. However, the underlying mechanisms remain unclear. We herein identified a quantitative trait locus, designated as q10.3, along with three lead single nucleotide polymorphisms (SNPs) located near a TPR gene (TETRATRICOPEPTIDE REPEAT), named CsTPR, through performing a genome-wide association study (GWAS) on 130 tea accessions. Integrated analysis of RNA-seq and ATAC-seq confirmed CsTPR as a droopiness-associated candidate gene at the transcriptional level. CsTPR was then proved to negatively regulate brassinosteroid-induced droopiness by using the CsTPR-silencing tea plant. Whole-genome sequencing (WGS) combined with genome walking further indicated that a single-base mutation (T–A) in the promoter of CsTPR. ChIP-seq revealed that this mutation occurred within the binding site, E-box, of CsBES1.2 on the CsTPR promoter. Notably, CsBES1.2 bound the E-box of CsTPR promoter to repress the expression of CsTPR, as demonstrated by chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), electrophoretic mobility shift assays (EMSA), and transient assays. The single-base mutation strengthened the inhibitory effect of CsBES1.2 on the expression of CsTPR via enhancing the binding affinity to the E-box. Altogether, our findings suggest that CsTPR negatively regulates droopiness in tea plants under the transcriptional repression of CsBES1.2 and that a single-base mutation within E-box amplifies the suppression of CsBES1.2 on the expression of CsTPR.

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