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In fleshy fruit, sugars and acids are central components of fruit flavor and quality. To date, the mechanisms underlying transcriptional regulation of sugar and acid during fruit development remain largely unknown. Here, we combined ATAC-seq with RNA-seq to investigate the genome-wide chromatin accessibility and to identify putative transcription factors related to sugar and acid accumulation during apple (Malus domestica) fruit development. By integrating the differentially accessible regions and differentially expressed genes, we generated a global data set of promoter-accessibility and expression-increased genes. Using this strategy, we constructed a transcriptional regulatory network enabling screening for key transcription factors and target genes involved in sugar and acid accumulation. Among these transcription factors, 5 fruit-specific DNA binding with one finger genes were selected to confirm their regulatory effects, and our results showed that they could affect sugar or acid concentration by regulating the expression of sugar or acid metabolism-related genes in apple fruits. Our transcriptional regulatory network provides a suitable platform to identify candidate genes that control sugar and acid accumulation. Meanwhile, our data set will aid in analyzing other characteristics of apple fruit that have not been illuminated previously. Overall, these findings support a better understanding of the regulatory dynamics during apple fruit development and lay a foundation for quality improvement of apple.
Assuntos
Frutas , Regulação da Expressão Gênica de Plantas , Malus , Açúcares , Malus/genética , Malus/metabolismo , Malus/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Açúcares/metabolismo , Redes Reguladoras de Genes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ácidos/metabolismo , Metabolismo dos Carboidratos/genéticaRESUMO
Malic acid is an important flavor determinant in apple (Malus domestica Borkh.) fruit. One known variation controlling malic acid is the A/G SNP in an aluminium-activated malate transporter gene (MdMa1). Nevertheless, there are still differences in malic acid content in apple varieties with the same Ma1 genotype (Ma1/Ma1 homozygous), such as 'Honeycrisp' (high malic acid content) and 'Qinguan' (low malic acid content), indicating that other loci may influence malic acid and fruit acidity. Here, the F1 hybrid generation of 'Honeycrisp' × 'Qinguan' was used to analyze quantitative trait loci (QTLs) for malic acid content. A major locus (Ma7) was identified on chromosome 13. Within this locus, a malate dehydrogenase gene, MDH1 (MdMa7), was the best candidate for further study. Subcellular localization suggested that MdMa7 encodes a cytosolic protein. Overexpression and RNAi of MdMa7 in apple fruit increased and decreased malic acid content, respectively. An insertion / deletion (indel) in the MdMa7 promoter was found to affect MdMa7 expression and malic acid content in both hybrids and other cultivated varieties. The insertion and deletion genotypes were designated as MA7 and ma7, respectively. The transcription factor MdbHLH74 was found to stimulate MdMa7 expression in the MA7 genotype but not in the ma7 genotype. Transient transformation of fruit showed that MdbHLH74 affected MdMa7 expression and malic acid content in 'Gala' (MA7/MA7) but not in 'Fuji' (ma7/ma7). Our results indicated that genetic variation in the MdMa7 (MDH1) promoter alters the binding ability of the transcription factor MdbHLH74, which alters MdMa7 (MDH1) transcription and the malic acid content in apple fruit, especially in Ma1/Ma1 homozygous accessions.
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Acidity is a key factor controlling fruit flavor and quality. In a previous study, combined transcriptome and methylation analyses identified a P3A-type ATPase from apple (Malus domestica), MdMa11, which regulates vacuolar pH when expressed in Nicotiana benthamiana leaves. In this study, the role of MdMa11 in controlling fruit acidity was verified in apple calli, fruits, and plantlets. In addition, we isolated an AP2 domain-containing transcription factor, designated MdESE3, based on yeast one-hybrid (Y1H) screening using the MdMa11 promoter as bait. A subcellular localization assay indicated that MdESE3 localized to the nucleus. Analyses of transgenic apple calli, fruits, and plantlets, as well as tomatoes, demonstrated that MdESE3 enhances fruit acidity and organic acid accumulation. Meanwhile, chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), luciferase (LUC) transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the ethylene-responsive element (ERE; 5'-TTTAAAAT-3') upstream of the MdMa11 transcription start site, thereby activating its expression. Furthermore, MdtDT, MdDTC2, and MdMDH12 expression increased in apple fruits and plantlets overexpressing MdESE3 and decreased in apple fruits and plantlets where MdESE3 was silenced. The ERE was found in MdtDT and MdMDH12 promoters, but not in the MdDTC2 promoter. The Y1H, LUC transactivation assays, and GUS reporter assays indicated that MdESE3 could bind to the MdtDT and MdMDH12 promoters and activate their expression. Our findings provide valuable functional validation of MdESE3 and its role in the transcriptional regulation of MdMa11, MdtDT, and MdMDH12 and malic acid accumulation in apple.
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In plants under drought stress, sugar content in roots increases, which is important for drought resistance. However, the molecular mechanisms for controlling the sugar content in roots during response to drought remain elusive. Here, we found that the MdDOF3-MdHT1.2 module-mediated glucose influx into the root is essential for drought resistance in apple (Malus × domestica). Drought induced glucose uptake from the rhizosphere and up-regulated the transcription of hexose transporter MdHT1.2. Compared with the wild-type plants, overexpression of MdHT1.2 promoted glucose uptake from the rhizosphere, thereby facilitating sugar accumulation in root and enhancing drought resistance, whereas silenced plants showed the opposite phenotype. Furthermore, ATAC-seq, RNA-seq and biochemical analysis demonstrated that MdDOF3 directly bound to the promoter of MdHT1.2 and was strongly up-regulated under drought. Overexpression of MdDOF3 in roots improved MdHT1.2-mediated glucose transport capacity and enhanced plant resistance to drought, but MdDOF3-RNAihr apple plants showed the opposite phenotype. Moreover, overexpression of MdDOF3 in roots did not attenuate drought sensitivity in MdHT1.2-RNAi plants, which was correlated with a lower glucose uptake capacity and glucose content in root. Collectively, our findings deciphered the molecular mechanism through which glucose uptake from the rhizosphere is mediated by MdDOF3-MdHT1.2, which acts to modulate sugar content in root and promote drought resistance.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Glucose , Malus , Proteínas de Plantas , Plantas Geneticamente Modificadas , Rizosfera , Malus/genética , Malus/metabolismo , Glucose/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Resistência à SecaRESUMO
Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Plantas Geneticamente Modificadas , Pyrus , Fatores de Transcrição , Xilema , Xilema/metabolismo , Xilema/genética , Pyrus/genética , Pyrus/metabolismo , Pyrus/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
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.
Assuntos
Temperatura Baixa , Frutas , Regulação da Expressão Gênica de Plantas , Malus , Proteínas de Plantas , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Frutas/genética , Frutas/metabolismo , Açúcares/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Plantas Geneticamente Modificadas , Metabolismo dos Carboidratos/genéticaRESUMO
Auxin plays important roles throughout plant growth and development. However, the mechanisms of auxin regulation of plant structure are poorly understood. In this study, we identified a transcription factor (TF) of the BARLEY B RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family in apple (Malus × domestica), MdBPC2. It was highly expressed in dwarfing rootstocks, and it negatively regulated auxin biosynthesis. Overexpression of MdBPC2 in apple decreased plant height, altered leaf morphology, and inhibited root system development. These phenotypes were due to reduced auxin levels and were restored reversed after exogenous indole acetic acid (IAA) treatment. Silencing of MdBPC2 alone had no obvious phenotypic effect, while silencing both Class I and Class II BPCs in apple significantly increased auxin content in plants. Biochemical analysis demonstrated that MdBPC2 directly bound to the GAGA-rich element in the promoters of the auxin synthesis genes MdYUC2a and MdYUC6b, inhibiting their transcription and reducing auxin accumulation in MdBPC2 overexpression lines. Further studies established that MdBPC2 interacted with the polycomb group (PcG) protein LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) to inhibit MdYUC2a and MdYUC6b expression via methylation of histone 3 lysine 27 (H3K27me3). Silencing MdLHP1 reversed the negative effect of MdBPC2 on auxin accumulation. Our results reveal a dwarfing mechanism in perennial woody plants involving control of auxin biosynthesis by a BPC transcription factor, suggesting its use for genetic improvement of apple rootstock.
Assuntos
Malus , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Malus/genética , Malus/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismoRESUMO
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.
Assuntos
Malus , Solanum lycopersicum , Malus/genética , Açúcares/metabolismo , Glucose/metabolismo , Solanum lycopersicum/genética , Vacúolos/metabolismo , Carboidratos , Regulação da Expressão Gênica de PlantasRESUMO
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.
Assuntos
Malus , Malus/genética , Malus/metabolismo , Frutas/genética , Frutas/metabolismo , Malatos/metabolismo , Locos de Características Quantitativas/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Plant roots can absorb sugars from the rhizosphere, which reduces the consumption of carbon derived from photosynthesis. However, the underlying mechanisms that roots use to control sugar absorption from soil are poorly understood. Here, we identified an apple (Malus × domestica Borkh.) hexose transporter, MdHT1.2, that functions on the root epidermis to absorb glucose (Glc) from the rhizosphere. Based on RNA-seq data, MdHT1.2 showed the highest expression level among 29 MdHT genes in apple roots. Biochemical analyses demonstrated that MdHT1.2 was mainly expressed in the epidermal cells of fine roots, and its protein was located on the plasma membrane. The roots of transgenic apple and Solanum lycopersicum lines overexpressing MdHT1.2 had an increased capability to absorb Glc when fed with [13C]-labeled Glc or 2-NBDG, whereas silencing MdHT1.2 in apple showed the opposite results. Further studies established that MdHT1.2-mediated Glc absorption from the rhizosphere changed the carbon assimilate allocation between apple shoot and root, which regulated plant growth. Additionally, a grafting experiment in tomato confirmed that increasing the Glc uptake capacity in the root overexpressing MdHT1.2 could facilitate carbohydrate partitioning to the fruit. Collectively, our study demonstrated that MdHT1.2 functions on the root epidermis to absorb rhizospheric Glc, which regulates the carbohydrate allocation for plant growth and fruit sugar accumulation.
Assuntos
Malus , Malus/metabolismo , Glucose/metabolismo , Rizosfera , Açúcares/metabolismo , Carbono/metabolismo , Raízes de Plantas/metabolismoRESUMO
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 AâT 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.
Assuntos
Malus , Malus/genética , Malus/metabolismo , Frutas/genética , Frutas/metabolismo , Malatos/metabolismo , Genótipo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
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.
Assuntos
Malus , beta-Frutofuranosidase , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo , Malus/genética , Malus/metabolismo , Peróxido de Hidrogênio/metabolismo , Família Multigênica , Filogenia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Fructokinase (FRK) activates fructose through phosphorylation, which sends the activated fructose into primary metabolism and regulates fructose signaling capabilities in plants. The apple (Malus × domestica) FRK gene MdFRK2 shows especially high affinity to fructose, and its overexpression decreases fructose levels in the leaves of young plants. However, in the current study of mature plants, fruits of transgenic apple trees overexpressing MdFRK2 accumulated a higher level of fructose than wild-type (WT) fruits (at both young and mature stages). Transgenic apple trees with high mRNA MdFRK2 expression showed no significant differences in MdFRK2 protein abundance or FRK enzyme activity compared to WT in mature leaves, young fruits, and mature fruits. Immunoprecipitation-mass spectrometry analysis identified an skp1, cullin, F-box (SCF) E3 ubiquitin ligase, calcyclin-binding protein (CacyBP), that interacted with MdFRK2. RNA-sequencing analysis provided evidence for ubiquitin-mediated post-transcriptional regulation of MdFRK2 protein for the maintenance of fructose homeostasis in mature leaves and fruits. Further analyses suggested an MdCacyBP-MdFRK2 regulatory module, in which MdCacyBP interacts with and ubiquitinates MdFRK2 to facilitate its degradation by the 26S proteasome, thus decreasing the FRK enzyme activity to elevate fructose concentration in transgenic apple trees. This result uncovered an important mechanism underlying plant fructose homeostasis in different organs through regulating the MdFRK2 protein level via ubiquitination and degradation. Our study provides usable data for the future improvement of apple flavor and expands our understanding of the molecular mechanisms underlying plant fructose content and signaling regulation.
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Malus , Malus/metabolismo , Proteína A6 Ligante de Cálcio S100/genética , Proteína A6 Ligante de Cálcio S100/metabolismo , Homeostase , Frutose , Açúcares/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismoRESUMO
Arbuscular mycorrhizal fungi (AMF) and plants form a symbiotic relationship that promotes plant growth and development. However, the regulatory mechanisms through which AMF promote plant growth and development are largely unexplored. In this study, the apple rootstock M26 was assessed physiologically, transcriptionally and metabolically when grown with and without AMF inoculation. AMF significantly promoted the number of lateral root (LR) increase and shoot elongation. Root transcriptomic and metabolic data showed that AMF promoted lateral root development mainly by affecting glucose metabolism, fatty acid metabolism, and hormone metabolism. Shoot transcriptomic and metabolic data showed that AMF promoted shoot elongation mainly by affecting hormone metabolism and the expression of genes associated with cell morphogenesis. To investigate whether shoot elongation is caused by root development, we analyzed the root/shoot dry weight ratio. There was a correlation between shoot growth and root development, but analysis of root and shoot metabolites showed that the regulation of AMF on plant shoot metabolites is independent of root growth. Our study bridged the gap in the field of growth and development related to AMF.
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The types and proportions of soluble sugar and organic acid in fruit significantly affect flavor quality. However, there are few reports on the crosstalk regulation between metabolism of organic acid and sugar in fruit. Here, we found that the overexpression of cytoplasmic malate dehydrogenase genes (MdcyMDHs) not only increased the malate content but also increased the sucrose concentration in transgenic apple calli and mature fruit. Enzyme activity assays indicated that the overexpression of MdcyMDH1 and MdcyMDH5 enhanced sucrose phosphate synthase (SPS) activity in transgenic materials. RNA-seq and expression analysis showed that the expression levels of SPS genes were up-regulated in MdcyMDH1-overexpressed apple fruit and MdcyMDH5-overexpressed apple calli. Further study showed that the inhibition of MdSPSB2 or MdSPSC2 expression in MdcyMDH1 transgenic fruit could reduce or eliminate, respectively, the positive effect of MdcyMDH1 on sucrose accumulation. Moreover, some starch cleavage-related genes (MdBAM6.1/6.2, MdBMY8.1/8.2, MdISA1) and the key gluconeogenesis-related phosphoenolpyruvate carboxykinase MdPEPCK1 gene were significantly up-regulated in the transcriptome differentially expressed genes of mature fruit overexpressing MdcyMDH1. These results indicate that alteration of malate metabolism mediated by MdcyMDH might regulate the expression of MdSPSs and SPS activity via affecting starch metabolism or gluconeogenesis, and thus accelerate sucrose synthesis and accumulation in fruit.
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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.
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Ascorbate (Asc) is an important antioxidant in plants and humans that plays key roles in various physiological processes. Understanding the regulation of Asc content in fruit plants is important for improving plant resiliency and optimizing Asc in food. Here, we found that both the transcript level and protein abundance of Asc Mannose pathway Regulator 1 Like 1 (MdAMR1L1) was negatively associated with Asc levels during the development of apple (Malus × domestica) fruit. The overexpression or silencing of MdAMR1L1 in apple indicated that MdAMR1L1 negatively regulated Asc levels. However, in the leaves of MdAMR1L1-overexpressing apple lines, the transcript levels of the Asc synthesis gene Guanosine diphosphate-mannose pyrophosphorylase MdGMP1 were increased, while its protein levels and enzyme activity were reduced. This occurred because the MdAMR1L1 protein interacted with MdGMP1 and promoted its degradation via the ubiquitination pathway to inhibit Asc synthesis at the post-translational level. MdERF98, an apple ethylene response factor, whose transcription was modulated by Asc level, is directly bound to the promoter of MdGMP1 to promote the transcription of MdGMP1. These findings provide insights into the regulatory mechanism of Asc biosynthesis in apples and revealed potential opportunities to improve fruit Asc levels.
Assuntos
Ácido Ascórbico/biossíntese , Ácido Ascórbico/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Malus/genética , Malus/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Células Cultivadas , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , GenótipoRESUMO
The content of organic acids greatly influences the taste and storage life of fleshy fruit. Our current understanding of the molecular mechanism of organic acid accumulation in apple (Malus domestica) fruit focuses on the aluminum-activated malate transporter 9/Ma1 gene. In this study, we identified a candidate gene, MdWRKY126, for controlling fruit acidity independent of Ma1 using homozygous recessive mutants of Ma1, namely Belle de Boskoop "BSKP" and Aifeng "AF." Analyses of transgenic apple calli and flesh and tomato (Solanum lycopersicum) fruit demonstrated that MdWRKY126 was substantially associated with malate content. MdWRKY126 was directly bound to the promoter of the cytoplasmic NAD-dependent malate dehydrogenase MdMDH5 and promoted its expression, thereby enhancing the malate content of apple fruit. In MdWRKY126 overexpressing calli, the mRNA levels of malate-associated transporters and proton pump genes also significantly increased, which contributed to the transport of malate accumulated in the cytoplasm to the vacuole. These findings demonstrated that MdWRKY126 regulates malate anabolism in the cytoplasm and coordinates the transport between cytoplasm and vacuole to regulate malate accumulation. Our study provides useful information to improve our understanding of the complex mechanism regulating apple fruit acidity.
Assuntos
Malus , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Fructose (Fru) content is a key determinant of fruit sweetness and quality. An F1 hybrid population of the apple cultivars 'Honeycrisp' × 'Qinguan' was used to investigate the quantitative trait locus (QTL) regions and genes controlling Fru content in fruit. A stable QTL on linkage group (LG) 01 in 'Honeycrisp' was detected on the single nucleotide polymorphism (SNP) genetic linkage maps. In this region, a sorbitol dehydrogenase (SDH) gene, MdSDH2, was detected and showed promoter variations and differential expression patterns between 'Honeycrisp' and 'Qinguan' fruits as well as their hybrids. A SNP variant (A/G) in the MdSDH2 promoter region (SDH2p-491) affected the binding ability of the transcription factor MdABI3, which can affect the expression of MdSDH2. Promoter sequences with an A nucleotide at SDH2p-491 had stronger binding affinity for MdABI3 than those with a G. Among 27 domesticated apple cultivars and wild relatives, this SNP (A/G) was associated with Fru content. Our results indicate that MdSDH2 can alter Fru content as the major regulatory gene and that ABA signaling might be involved in Fru content accumulation in apple fruit.
Assuntos
Malus , Frutose/metabolismo , Frutas/metabolismo , L-Iditol 2-Desidrogenase/genética , Malus/genética , Malus/metabolismo , Regiões Promotoras Genéticas/genética , Sorbitol/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.