A single-base mutation in promoter of CsTPR enhances the negative regulation on mechanical-related leaf drooping in tea plants

Abstract

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.

Acknowledgements

This work was supported by the grants from Zhejiang Provincial Natural Science Foundation of China (LQ23C160010), Jiangsu Seed Industry Revitalization Project JBGS[2021]081, Fundamental Research Fund for Tea Research Institute of the Chinese Academy of Agricultural Sciences (grant number 1610212022002), Jiangxi Province Talent Plan (jxsq2023102020), China Agriculture Research System of MOF and MARA (CARS-19), Yunnan Province of Science and Technology Department (202449CE340010), and National Key Laboratory for Tea Plant Germplasm Innovation and Resource Utilization (SQ2024SKL03104).

Author contributions

H.L. conceived, designed, and performed all the experiments. L.D. and C.T. performed most of experiments. J.J. performed some experiments. J.M. and J.C. performed GWAS and analyzed data. W.H. provided direction of researches and managed planting for resources. M.Y. and L.C. designed experiments and provided direction of researches. H.L. wrote the manuscript. All authors approved the manuscript.

Data availability

The genomic and sequencing data associated with this study are available in public repositories as follows: RNA-seq and ChIP-seq data are accessible under NCBI accession PRJNA1075292, while WGS and ATAC-seq data can be retrieved using accession numbers PRJNA1206779 and PRJNA1207240, respectively. The gene sequences of CsTPR (CSS0022606) and CsBES1.2 (CSS0038858) are available in the tea genome database. Additionally, whole-genome sequencing data for 130 tea accessions used in GWAS analysis are publicly accessible through TeaGVD (http://www.teaplant.top/teagvd) and listed in Supplemental Table S1.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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