The MdCBF1/2-MdTST1/2 module regulates sugar accumulation in response to low temperature in apple.

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.

Transcriptional repression of MdMa1 by MdMYB21 in Ma locus decreases malic acid content in apple fruit.

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.

Allelic variation of MdMYB123 controls malic acid content by regulating MdMa1 and MdMa11 expression in apple.

Acidity is a key determinant of fruit organoleptic quality. Here, a candidate gene for fruit acidity, designated MdMYB123, was identified from a comparative transcriptome study of two Ma1Ma1 apple (Malus domestica) varieties, "Qinguan (QG)" and "Honeycrisp (HC)" with different malic acid content. Sequence analysis identified an A→T SNP, which was located in the last exon, resulting in a truncating mutation, designated mdmyb123. This SNP was significantly associated with fruit malic acid content, accounting for 9.5% of the observed phenotypic variation in apple germplasm. Differential MdMYB123- and mdmyb123-mediated regulation of malic acid accumulation was observed in transgenic apple calli, fruits, and plantlets. Two genes, MdMa1 and MdMa11, were up- and down-regulated in transgenic apple plantlets overexpressing MdMYB123 and mdmyb123, respectively. MdMYB123 could directly bind to the promoter of MdMa1 and MdMa11, and induce their expression. In contrast, mdmyb123 could directly bind to the promoters of MdMa1 and MdMa11, but with no transcriptional activation of both genes. In addition, gene expression analysis in 20 different apple genotypes based on SNP locus from "QG" × "HC" hybrid population confirmed a correlation between A/T SNP with expression levels of MdMa1 and MdMa11. Our finding provides valuable functional validation of MdMYB123 and its role in the transcriptional regulation of both MdMa1 and MdMa11, and apple fruit malic acid accumulation.

Genome-wide identification, characterization and evolutionary dynamic of invertase gene family in apple, and revealing its roles in cold tolerance.

Invertases are ubiquitous enzymes that catalyze the unalterable cleavage of sucrose into glucose and fructose, and are crucially involved in plant growth, development and stress response. In this study, a total of 17 putative invertase genes, including 3 cell wall invertases, 3 vacuolar invertases, and 11 neutral invertases were identified in apple genome. Subcellular localization of MdNINV7 and MdNINV11 indicated that both invertases were located in the cytoplasm. Comprehensive analyses of physicochemical properties, chromosomal localization, genomic characterization, and gene evolution of MdINV family were conducted. Gene duplication revealed that whole-genome or segmental duplication and random duplication might have been the major driving force for MdINVs expansion. Selection index values, ω, showed strong evidence of positive selection signatures among the INV clusters. Gene expression analysis indicated that MdNINV1/3/6/7 members are crucially involved in fruit development and sugar accumulation. Similarly, expression profiles of MdCWINV1, MdVINV1, and MdNINV1/2/7/11 suggested their potential roles in response to cold stress. Furthermore, overexpression of MdNINV11 in apple calli at least in part promoted the expression of MdCBF1-5 and H2O2 detoxification in response to cold. Overall, our results will be useful for understanding the functions of MdINVs in the regulation of apple fruit development and cold stress response.

The SnRK2.3-AREB1-TST1/2 cascade activated by cytosolic glucose regulates sugar accumulation across tonoplasts in apple and tomato.

Soluble sugars are the core components of fruit quality, and the degree of sugar accumulation is largely determined by tonoplast-localized sugar transporters. We previously showed that two classes of tonoplast sugar transporters, MdERDL6 and MdTST1/2, coordinately regulate sugar accumulation in vacuoles. However, the mechanism underlying this coordination remains unknown. Here we discovered that two transcription factors, MdAREB1.1/1.2, regulate MdTST1/2 expression by binding their promoters in apple. The enhanced MdAREB1.1/1.2 expression in MdERDL6-1-overexpression plants resulted in an increase in MdTST1/2 expression and sugar concentration. Further studies established that MdSnRK2.3, whose expression could be regulated by expressing MdERDL6-1, could interact with and phosphorylate MdAREB1.1/1.2, thereby promoting the MdAREB1.1/1.2-mediated transcriptional activation of MdTST1/2. Finally, the orthologous SlAREB1.2 and SlSnRK2.3 exhibited similar functions in tomato fruit as in their apple counterparts. Together, our findings provide insights into the regulatory mechanism of tonoplast sugar transport exerted by SnRK2.3-AREB1-TST1/2 for fruit sugar accumulation.

Overexpression of apple Ma12, a mitochondrial pyrophosphatase pump gene, leads to malic acid accumulation and the upregulation of malate dehydrogenase in tomato and apple calli.

Acidity is an important factor influencing the organoleptic quality of apple fruits. In this study, an apple pyrophosphate-energized proton pump (PEPP) gene was isolated and designated MdMa12. On the basis of a phylogenetic analysis in Rosaceae species, PEPP genes were divided into three groups, with apple PEPP genes most closely related to pear PEPP genes. Gene expression analysis revealed that high malic acid content was generally accompanied by high MdMa12 expression levels. Moreover, MdMa12 was mainly expressed in the fruit. A subcellular localization analysis suggested that MdMa12 is a mitochondrial protein. The ectopic expression and overexpression of MdMa12 in "Micro-Tom" tomato and apple calli, respectively, increased the malic acid content. One (MDH12) of four malate dehydrogenase genes highly expressed in transgenic apple calli was confirmed to encode a protein localized in mitochondria. The overexpression of MDH12 increased the malate content in apple calli. Furthermore, MdMa12 overexpression increased MdDTC1, MdMa1, and MdMa10 expression levels, which were identified to transport malate. These findings imply that MdMa12 has important functions related to apple fruit acidity. Our study explored the regulatory effects of mitochondria on the complex mechanism underlying apple fruit acidity.

Combined Profiling of Transcriptome and DNA Methylome Reveal Genes Involved in Accumulation of Soluble Sugars and Organic Acid in Apple Fruits.

Organic acids and soluble sugars are the major determinants of fruit organoleptic quality. Additionally, DNA methylation has crucial regulatory effects on various processes. However, the epigenetic modifications in the regulation of organic acid and soluble sugar accumulation in apple fruits remain uncharacterized. In this study, DNA methylation and the transcriptome were compared between 'Honeycrisp' and 'Qinguan' mature fruits, which differ significantly regarding soluble sugar and organic acid contents. In both 'Honeycrisp' and 'Qinguan' mature fruits, the CG context had the highest level of DNA methylation, and then CHG and CHH contexts. The number and distribution of differentially methylated regions (DMRs) varied among genic regions and transposable elements. The DNA methylation levels in all three contexts in the DMRs were significantly higher in 'Honeycrisp' mature fruits than in 'Qinguan' mature fruits. A combined methylation and transcriptome analysis revealed a negative correlation between methylation levels and gene expression in DMRs in promoters and gene bodies in the CG and CHG contexts and in gene bodies in the CHH context. Two candidate genes (MdTSTa and MdMa11), which encode tonoplast-localized proteins, potentially associated with fruit soluble sugar contents and acidity were identified based on expression and DNA methylation levels. Overexpression of MdTSTa in tomato increased the fruit soluble sugar content. Moreover, transient expression of MdMa11 in tobacco leaves significantly decreased the pH value. Our results reflect the diversity in epigenetic modifications influencing gene expression and will facilitate further elucidating the complex mechanism underlying fruit soluble sugar and organic acid accumulation.