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' x '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.

Calcyclin-binding protein-promoted degradation of MdFRUCTOKINASE2 regulates sugar homeostasis in apple.

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 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.

Transcriptomics and metabolomics reveal effect of arbuscular mycorrhizal fungi on growth and development of apple plants.

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.

Malate metabolism mediated by the cytoplasmic malate dehydrogenase gene MdcyMDH affects sucrose synthesis in apple fruit.

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.

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.

MdWRKY126 modulates malate accumulation in apple fruit by regulating cytosolic malate dehydrogenase (MdMDH5).

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.

F-box protein MdAMR1L1 regulates ascorbate biosynthesis in apple by modulating GDP-mannose pyrophosphorylase.

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.

Variation in the promoter of the sorbitol dehydrogenase gene MdSDH2 affects binding of the transcription factor MdABI3 and alters fructose content in apple fruit.

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.

MdFRK2-mediated sugar metabolism accelerates cellulose accumulation in apple and poplar.

BACKGROUND: Cellulose is not only a common component in vascular plants, but also has great economic benefits for paper, wood, and industrial products. In addition, its biosynthesis is highly regulated by carbohydrate metabolism and allocation in plant. MdFRK2, which encodes a key fructokinase (FRK) in apple, showed especially high affinity to fructose and regulated carbohydrate metabolism. RESULTS: It was observed that overexpression of MdFRK2 in apple decreased sucrose (Suc) and fructose (Fru) with augmented FRK activity in stems, and caused the alterations of many phenotypic traits that include increased cellulose content and an increase in thickness of the phloem region. To further investigate the involved mechanisms, we generated FRK2-OE poplar lines OE#1, OE#4 and OE#9 and discovered (1) that overexpression of MdFRK2 resulted in the huge increased cellulose level by shifting the fructose 6-phosphate or glucose 6-phsophate towards UDPG formation, (2) a direct metabolic pathway for the biosynthesis of cellulose is that increased cleavage of Suc into UDP-glucose (UDPG) for cellulose synthesis via the increased sucrose synthase (SUSY) activity and transcript levels of PtrSUSY1, (3) that the increased FRK activity increases the sink strength overall so there is more carbohydrate available to fuel increased cambial activity and that resulted in more secondary phloem. These results demonstrated that MdFRK2 overexpression would significantly changes the photosynthetic carbon flux from sucrose and hexose to UDPG for increased cellulose synthesis. CONCLUSIONS: The present data indicated that MdFRK2 overexpression in apple and poplar changes the photosynthetic carbon flux from sucrose and hexose to UDPG for stem cellulose synthesis. A strategy is proposed to increase cellulose production by regulating sugar metabolism as a whole.

Unraveling a genetic roadmap for improved taste in the domesticated apple.

Although taste is an important aspect of fruit quality, an understanding of its genetic control remains elusive in apple and other fruit crops. In this study, we conducted genomic sequence analysis of 497 Malus accessions and revealed erosion of genetic diversity caused by apple breeding and possible independent domestication events of dessert and cider apples. Signatures of selection for fruit acidity and size, but not for fruit sugar content, were detected during the processes of both domestication and improvement. Furthermore, we found that single mutations in major genes affecting fruit taste, including Ma1, MdTDT, and MdSOT2, dramatically decrease malate, citrate, and sorbitol accumulation, respectively, and correspond to important domestication events. Interestingly, Ma1 was identified to have pleiotropic effects on both organic acid content and sugar:acid ratio, suggesting that it plays a vital role in determining fruit taste. Fruit taste is unlikely to have been negatively affected by linkage drag associated with selection for larger fruit that resulted from the pyramiding of multiple genes with minor effects on fruit size. Collectively, our study provides new insights into the genetic basis of fruit quality and its evolutionary roadmap during apple domestication, pinpointing several candidate genes for genetic manipulation of fruit taste in apple.

Nitrogen Levels Regulate Sugar Metabolism and Transport in the Shoot Tips of Crabapple Plants.

To comprehensively understand the responses of carbohydrate metabolism and transport to different levels of nitrogen supply in growing shoot tips of crabapple (Malus hupehensis Rehd), enzyme activities and related genes involved in the sugar metabolism pathway were investigated. The nitrogen and chlorophyll content of plants increased with increasing nitrogen supply. High nitrogen application increased the net photosynthesis rate and the growth rate of shoot tips but decreased the synthesis capability of sucrose and sorbitol in mature leaves. However, the shoot tips of plants under high-nitrogen treatment had higher contents of sucrose and sorbitol than did those under low-nitrogen treatment, while the activity of sucrose phosphate synthase and aldose-6-phosphate was increased and the transporters MdSOT and MdSUT were up-regulated. Moreover, the activities of enzymes involved in sucrose and hexose metabolism (including sucrose synthase, fructokinase, and hexokinase) were enhanced in the shoot tips of plants under high-nitrogen conditions, and the expression levels of MdSUSY3 and MdHK1 were significantly up-regulated. These findings indicate that a high nitrogen supply increases the metabolic capacity of assimilatory substances in shoot tips, accelerates the efficiency of sugar utilization and eventually leads to a rapid increase in the growth of shoot tips. Our results highlight that high nitrogen increases the capacity of sugar unloading and metabolic utilization in growing shoot tissues.

MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato.

Sugar transport across tonoplasts is essential for maintaining cellular sugar homeostasis and metabolic balance in plant cells. It remains unclear, however, how this process is regulated among different classes of sugar transporters. Here, we identified a tonoplast H+/glucose symporter, MdERDL6-1, from apples, which was highly expressed in fruits and exhibited expression patterns similar to those of the tonoplast H+/sugar antiporters MdTST1 and MdTST2. Overexpression of MdERDL6-1 unexpectedly increased not only glucose (Glc) concentration but also that of fructose (Fru) and sucrose (Suc) in transgenic apple and tomato leaves and fruits. RNA sequencing (RNA-seq) and expression analyses showed an up-regulation of TST1 and TST2 in the transgenic apple and tomato lines overexpressing MdERDL6-1 Further studies established that the increased sugar concentration in the transgenic lines correlated with up-regulation of TST1 and TST2 expression. Suppression or knockout of SlTST1 and SlTST2 in the MdERDL6-1-overexpressed tomato background reduced or abolished the positive effect of MdERDL6-1 on sugar accumulation, respectively. The findings demonstrate a regulation of TST1 and TST2 by MdERDL6-1, in which Glc exported by MdERDL6-1 from vacuole up-regulates TST1 and TST2 to import sugars from cytosol to vacuole for accumulation to high concentrations. The results provide insight into the regulatory mechanism of sugar accumulation in vacuoles mediated by the coordinated action of two classes of tonoplast sugar transporters.

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.

Direct and Bicarbonate-Induced Iron Deficiency Differently Affect Iron Translocation in Kiwifruit Roots.

Bicarbonate-induced iron (Fe) deficiency (+Bic) is frequently observed in kiwifruit orchards, but more research attention has been paid to direct Fe deficiency (-Fe) in plants, including kiwifruit. Here we compared the differences of kiwifruit plants between -Fe and +Bic in: (1) the traits of 57Fe uptake and translocation within plants, (2) Fe forms in roots, and (3) some acidic ions and metabolites in roots. The concentration of 57Fe derived from nutrient solution (57Fedfs) in roots was less reduced in +Bic than -Fe treatment, despite similar decrease in shoots of both treatments. +Bic treatment increased 57Fedfs distribution in fine roots but decreased it in new leaves and stem, thereby displaying the inhibition of 57Fedfs translocation from roots to shoots and from fine roots to xylem of coarse roots. Moreover, +Bic imposition induced the accumulation of water-soluble Fe and apoplastic Fe in roots. However, the opposite was observed in -Fe-treated plants. Additionally, the cell wall Fe and hemicellulose Fe in roots were less reduced by +Bic than -Fe treatment. +Bic treatment also triggered the reduction in H+ extrusion and the accumulation of NH4+, succinic acid, and some amino acids in roots. These results suggest that, contrary to -Fe, +Bic treatment inhibits Fe translocation to shoots by accumulating water-soluble and apoplastic Fe and slowing down the release of hemicellulose Fe in the cell wall in kiwifruit roots, which may be related to the decreased H+ extrusion and the imbalance between C and N metabolisms.

Ectopic expression of apple hexose transporter MdHT2.2 reduced the salt tolerance of tomato seedlings with decreased ROS-scavenging ability.

Salt is one of the main stresses that limit plant growth, especially at the seedling stage, reducing crop production and severely impacting food security. However, the relationship between salt stress and sugar content regulated by sugar transporters remains unknown. Here, we investigated the salt tolerance of transgenic tomato seedlings ectopically expressing MdHT2.2, which is a fructose and glucose/H+ symporter located on the plasma membrane in apple. Although the contents of fructose, glucose and sucrose in the leaves of seedlings ectopically expressing MdHT2.2 obviously increased compared with those of WT seedlings, the transgenic seedlings were significantly less tolerance to salt stress. Under salt stress, the SlSOS1/2 and SlNHX1 genes were highly expressed, and the accumulation of Na+ was lower in the transgenic seedlings than in WT, however, ROS accumulated to a greater degree in the former, and the ROS-scavenging-related enzyme activities and AsA content were lower in the transgenic seedlings than WT. Taken together, these results indicated that the relatively low salt tolerance of the MdHT2.2 transgenic seedlings was related with the accumulation of ROS, which was caused by reduced ROS-scavenging ability. Our results offer proof that changes in sugar content caused by sugar transporters are related to salt tolerance, and provide new insight into the regulation of sugar content, quality improvement and stress tolerance.

Transcriptomic and Metabolic Analyses Provide New Insights into the Apple Fruit Quality Decline during Long-Term Cold Storage.

Long-term low-temperature conditioning (LT-LTC) decreases apple fruit quality, but the underlying physiological and molecular basis is relatively uncharacterized. We identified 12 clusters of differentially expressed genes (DEGs) involved in multiple biological processes (i.e., sugar, malic acid, fatty acid, lipid, complex phytohormone, and stress-response pathways). The expression levels of genes in sugar pathways were correlated with decreasing starch levels during LT-LTC. Specifically, starch-synthesis-related genes (e.g., BE, SBE, and GBSS genes) exhibited downregulated expression, whereas sucrose-metabolism-related gene expression levels were up- or downregulated. The expression levels of genes in the malic acid pathway (ALMT9, AATP1, and AHA2) were upregulated, as well as the content of malic acid in apple fruit during LT-LTC. A total of 151 metabolites, mainly related to amino acids and their isoforms, amines, organic acids, fatty acids, sugars, and polyols, were identified during LT-LTC. Additionally, 35 organic-acid-related metabolites grouped into three clusters, I (3), II (22), and III (10), increased in abundance during LT-LTC. Multiple phytohormones regulated the apple fruit chilling injury response. The ethylene (ET) and abscisic acid (ABA) levels increased at CS2 and CS3, and jasmonate (JA) levels also increased during LT-LTC. Furthermore, the expression levels of genes involved in ET, ABA, and JA synthesis and response pathways were upregulated. Finally, some key transcription factor genes (MYB, bHLH, ERF, NAC, and bZIP genes) related to the apple fruit cold acclimation response were differentially expressed. Our results suggest that the multilayered mechanism underlying apple fruit deterioration during LT-LTC is a complex, transcriptionally regulated process involving cell structures, sugars, lipids, hormones, and transcription factors.

Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit.

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high 14 C-fructose and 14 C-glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up-regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5-edited mutant of the MdHT2.2-expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.

Comparative Proteomic Analysis Reveals Key Proteins Linked to the Accumulation of Soluble Sugars and Organic Acids in the Mature Fruits of the Wild Malus Species.

Soluble sugars and organic acids are the main determinants of fruit organoleptic quality. To investigate the genes responsible for the soluble sugar and organic acid contents of apple fruits, a label-free proteomic analysis involving liquid chromatography (LC)-mass spectrometry (MS)/MS was conducted with the fruits of two Malus species, M. sargentii and M. niedzwetzkyana, which exhibit significant differences in soluble sugar and organic acid contents. A total of 13,036 unique peptides and 1,079 differentially-expressed proteins were identified. To verify the LC-MS/MS results, five candidate proteins were further analyzed by parallel reaction monitoring. The results were consistent with the LC-MS/MS data, which confirmed the reliability of the LC-MS/MS analysis. The functional annotation of the differentially-expressed proteins, based on the gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, revealed that they were mainly related to biological processes and cellular components. Additionally, the main enriched KEGG pathways were related to metabolic processes. Moreover, 31 proteins involved in soluble sugar metabolism, organic acid metabolism, and H+-transport were identified. The results of this study may be useful for the comprehensive characterization of the complex mechanism regulating apple fruit-soluble sugar and organic acid contents.

Melatonin-Mediated Sugar Accumulation and Growth Inhibition in Apple Plants Involves Down-Regulation of Fructokinase 2 Expression and Activity.

Melatonin has been reported to play roles in regulating carbohydrate levels and plant growth. However, little is known about the exact mechanism by which melatonin regulates sugar levels and growth in plants. In this study, it was found that high levels of melatonin inhibited the growth of wild-type (WT) apple plants and induced significant accumulations of fructose, glucose, and sucrose in apple leaves, while MdFRK2 expression was significantly downregulated. MdFRK2 promoter transiently expressed in tobacco leaves further supported that the expression of MdFRK2 could be inhibited by exogenous melatonin. After applying exogenous melatonin, the suppression of MdFRK2 expression was significantly rescued in transgenic apples overexpressing MdFRK2 via the 35S promoter. Fructose, glucose, and sucrose concentrations increased less as compared to WT apple plants. Wild-type plants showed a stunted phenotype 21 days after melatonin treatment, while MdFRK2-overexpressing plants exhibited slightly inhibited growth, indicating that the downregulated MdFRK2 expression in response to melatonin was involved in melatonin-mediated growth inhibition. Taken together, these results demonstrate the involvement of MdFRK2 in melatonin-induced sugar accumulation and growth inhibition. Our findings shed light on the roles played by MdFRK2 in connecting melatonin action and plant growth.