Promoter variation affects binding affinity of the transcription factor MdWRKY20 to the Cell Wall Invertase 1 gene and decreases fructose content in apple fruit

Abstract

Apple sweetness is primarily attributed to the high content and perceived sweet taste of fructose. A previous study used an F1 hybrid population of Malus × domestica [‘Honeycrisp’ (HC) × ‘Qinguan’ (QG) (2n = 34)] to identify quantitative trait loci (QTLs) for fructose content in fruit, revealing a stable QTL on linkage group (LG) 03 in the HC genetic map. In this study, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses of genes within this interval in combination with RNA-sequencing identified a cell wall invertase gene MdCWINV1, whose expression was highly associated with the dynamic changes in fructose content in parental fruits. The coding sequences were conserved between the two cultivars, while the promoters carried 73 single nucleotide polymorphisms (SNPs). Based on transcriptional regulatory element prediction, a unique SNP, CWINV1pro-1080 (A/C), located at −1080 bp upstream of the ATG start codon in the HC-P1 haplotype, was identified and predicted to affect the binding of the transcription factor MdWRKY20. β-glucuronidase (GUS) assays, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR), dual-luciferase assays, and genetic transformation confirmed that MdWRKY20 specifically binds to the CWINV1pro-1080 (A) haplotype and significantly suppresses MdCWINV1 expression, reduces CWINV activity, and consequently decreases fructose accumulation. This study elucidated the functional role of MdCWINV1 as a key gene regulating fructose content and clarified how natural mutations in its promoter influence gene expression and sugar composition.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 32102330). We are grateful to Dr. J. Zhang and Miss W. Cao (Horticulture Science Research Center, Northwest A&F University, Yangling, China) for providing professional technical assistance with GC-MS analysis.

Author contributions

M.L., F.M., and Z.W. designed the research; Z.W., C.Z., and N.Y. performed the research; Z.W., C.Z., J.H., and M.L. analyzed the data; Z.W. and C.Z. wrote the paper; and M.L. and Z.W. supervised the research.

Data availability

All relevant data supporting our findings are available in the manuscript and Supplementary Materials. RNA sequencing data have been deposited in NCBI under accession number PRJNA1344609.

Conflicts of interest statement

The authors declare that they have no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

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