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SUMMARY: Soluble sugar content is a key component in controlling fruit flavor, and its accumulation in fruit is largely determined by sugar metabolism and transportation. When the diurnal temperature range is greater, the fleshy fruits accumulated more soluble sugars and become more sweeter. However, the molecular mechanism underlying this response remains largely unknown. In this study, we verified that low‐temperature treatment promoted soluble sugar accumulation in apple fruit and found that this was due to the upregulation of the Tonoplast Sugar Transporter genes MdTST1/2. A combined strategy using assay for transposase‐accessible chromatin (ATAC) sequencing and gene expression and cis‐acting elements analyses, we identified two C‐repeat Binding Factors, MdCBF1 and MdCBF2, that were induced by low temperature and that might be upstream transcription factors of MdTST1/2. Further studies established that MdCBF1/2 could bind to the promoters of MdTST1/2 and activate their expression. Overexpression of MdCBF1 or MdCBF2 in apple calli and fruit significantly upregulated MdTST1/2 expression and increased the concentrations of glucose, fructose, and sucrose. Suppression of MdTST1 and/or MdTST2 in an MdCBF1/2‐overexpression background abolished the positive effect of MdCBF1/2 on sugar accumulation. In addition, simultaneous silencing of MdCBF1/2 downregulated MdTST1/2 expression and apple fruits failed to accumulate more sugars under low‐temperature conditions, indicating that MdCBF1/2‐mediated sugar accumulation was dependent on MdTST1/2 expression. Hence, we concluded that the MdCBF1/2‐MdTST1/2 module is crucial for sugar accumulation in apples in response to low temperatures. Our findings provide mechanistic components coordinating the relationship between low temperature and sugar accumulation as well as new avenues to improve fruit quality. Significance Statement: This study showed that low temperature induced the expression levels of the transcription factors genes MdCBF1/2 in apple, which could bind to the promoters of the tonoplast sugar transporter genes MdTST1/2 and activate their expression, leading to Glc, Fru, and Suc accumulation at high concentrations in the vacuoles. [ABSTRACT FROM AUTHOR]

SUMMARY: Malic acid is a major organic acid component of apples and a crucial determinant of fruit organoleptic quality. A candidate gene for malic acid content, designated MdMa1, was previously identified in the Ma locus, which is a major quantitative trait locus (QTL) for apple fruit acidity located on the linkage group 16. Region‐based association mapping to detect candidate genes in the Ma locus identified MdMa1 and an additional MdMYB21 gene putatively associated with malic acid. MdMYB21 was significantly associated with fruit malic acid content, accounting for ~7.48% of the observed phenotypic variation in the apple germplasm collection. Analyses of transgenic apple calli, fruits and tomatoes demonstrated that MdMYB21 negatively regulated malic acid accumulation. The apple fruit acidity‐related MdMa1 and its tomato ortholog, SlALMT9, exhibited lower expression profiles in apple calli, mature fruits and tomatoes in which MdMYB21 was overexpressed, compared with their corresponding wild‐type variety. MdMYB21 directly binds to the MdMa1 promoter and represses its expression. Interestingly, a 2‐bp variation in the MdMYB21 promoter region altered its expression and regulation of its target gene, MdMa1, expression. Our findings not only demonstrate the efficiency of integrating QTL and association mapping in the identification of candidate genes controlling complex traits in apples, but also provide insights into the complex regulatory mechanism of fruit malic acid accumulation. Significance Statement: Fruit acidity is a complex quantitative trait that is regulated by one or more major and/or numerous minor genes. Region‐based association mapping was performed, and identified two candidate genes, MdMa1 and MdMYB21, which were associated with malic acid content in the Ma locus at linkage group (LG)16. Furthermore, MdMYB21 could negatively regulate malic acid accumulation in apple fruits by repressing the MdMa1 expression. [ABSTRACT FROM AUTHOR]