Integrative analysis of genome-wide association studies of polyphenols in apple fruits identifies the MdDof2.4-MdPAT10 module that promotes procyanidin accumulation

Abstract

Polyphenols represent a significant class of nutrients in apples, contributing to human health and well-being. Among these, procyanidins stand out as the most prevalent polyphenolic compounds in apple fruits. These compounds are abundant in wild apples and generally low in modern apple cultivars. Therefore, it is crucial to identify and recover genetically lost genes that regulate polyphenol accumulation in order to improve the apple quality. To achieve this, we conducted a genome-wide association study (GWAS) on 15 traits related to polyphenol content, utilizing 134 Malus accessions. We identified 1204 marker-trait associations (MTAs) and 840 candidate genes, including known polyphenol biosynthetic and regulatory genes, such as MYB7, TT4, and HCT/HQT. Notably, we pinpointed a protein S-acyl transferase 10 (PAT10), which is significantly associated with procyanidin content. Through experiments with transgenic calli, we determined that apple (Malus domestica) MdPAT10 positively regulated procyanidin accumulation. Furthermore, we identified a 51-bp insertion variant (In-868) on the promoter of the PAT10, which influences its expression. Both a yeast one-hybrid (Y1H) assay and an electrophoretic mobility shift assay (EMSA) revealed that MdDof2.4 was able to bind to the promoter of MdPAT10 containing In-868 (MdPAT10_pro^In-868), but not to the promoter of MdPAT10 without In-868 (MdPAT10_pro). Moreover, MdDof2.4 promoted MdPAT10 (with MdPAT10_pro^In-868) expression and increased procyanidin accumulation in fruits. Overall, our results enhance the understanding of the biosynthetic regulation of apple polyphenols and provide a theoretical foundation and genetic resources for breeding apple varieties with optimal polyphenol content.

Acknowledgements

We would like to thank Dr. Chunxiang You from Shandong Agricultural University for providing wild-type ‘Orin’ calli, Dr. Jing Zhang from Horticulture Science Research Center, Northwest A&F University (NWAFU), for providing professional technical assistance, and Arij Khalil from Department of Horticulture, Ghazi University, for the language modifications. We also thank the High-Performance Computing (HPC) platform of NWAFU for providing computing resources. This work was supported by the National Natural Science Foundation of China (32402541), the National Science Fund for Distinguished Young Scholars (32325045), China Postdoctoral Science Foundation (2024M752635), Shaanxi Postdoctoral Research Project (2023BSHEDZZ123), the Key S&T Special Projects of Shaanxi Province, China (2020zdzx03-01-02), and Chinese Universities Scientific Fund (2452023067).

Author contributions

Q.G. and P.Y. conceived and supervised the project; F.M. provided the Malus samples. ZX.L., C.G., Y.C., and X.Z. performed bioinformatics and statistical analyses; ZX.L., C.G., T.F., Y.C., ZY.L., L.L., Q.Q., M.C., and C.N. conducted the experiments. ZX.L., C.G., and Q.G. wrote the manuscript. All authors read and approved of the content.

Data availability statement

The raw whole-genome resequencing data of 134 Malus accessions were retrieved from the NCBI Sequence Read Archive (SRA) with the accession number PRJNA728537, and details in Table S1. Sequence data used in this article can be found in GenBank under the following accession numbers: XM_008339732.3 (MdPAT10); PQ100214 (MdDof2.4); XM_008354691.3 (MdMYB7).

Conflict of Interests

The authors declare no competing interests.

Supplementary information

Supplementary data is available at Horticulture Research online.

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