A Functional InDel in the WRKY10 Promoter Controls the Degree of Flesh Red Pigmentation in Apple.

MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red-flesh phenotype due to a minisatellite insertion in its R6 promoter, but R6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull-down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB-bHLH-WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163-bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R6 by structural variation analysis, contains a typical W-box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R6 background. These findings reveal a novel regulatory role of the WRKY-containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants.

Hypermethylation in the promoter regions of flavonoid pathway genes is associated with skin color fading during 'Daihong' apple fruit development.

Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar 'Daihong' (DH) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1, MdCHS, MdANS, MdUFGT, MdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5' and 3' flanking regions of genes and transposable elements (TEs), and in TE bodies in all CG, CHG and CHH contexts, especially the mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1, MdROS1, and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHS, MdCHI, MdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHS, MdCHI, MdANR, and MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin. Treatment with the DNA demethylation agent 5-aza-2'-deoxycytidine verified the negative correlation between DNA methylation and gene expression within the flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during DH apple development.

Mapping and spatiotemporal dynamics of land-use and land-cover change based on the Google Earth Engine cloud platform from Landsat imagery: A case study of Zhoushan Island, China.

Land resources are an essential foundation for socioeconomic development. Island land resources are limited, the type changes are particularly frequent, and the environment is fragile. Therefore, large-scale, long-term, and high-accuracy land-use classification and spatiotemporal characteristic analysis are of great significance for the sustainable development of islands. Based on the advantages of remote sensing indices and principal component analysis in accurate classification, and taking Zhoushan Archipelago, China, as the study area, in this work long-term satellite remote sensing data were used to perform land-use classification and spatiotemporal characteristic analysis. The classification results showed that the land-use types could be exactly classified, with the overall accuracy and Kappa coefficient greater than 94% and 0.93, respectively. The results of the spatiotemporal characteristic analysis showed that the built-up land and forest land areas increased by 90.00 km2 and 36.83 km2, respectively, while the area of the cropland/grassland decreased by 69.77 km2. The areas of the water bodies, tidal flats, and bare land exhibited slight change trends. The spatial coverage of Zhoushan Island continuously expanded toward the coast, encroaching on nearby sea areas and tidal flats. The cropland/grassland was the most transferred-out area, at up to 108.94 km2, and built-up land was the most transferred-in areas, at up to 73.31 km2. This study provides a data basis and technical support for the scientific management of land resources.

Distribution, source, and contamination assessment of heavy metals in surface sediments of the Zhifu Bay in northern China.

In this study, 30 surface sediment samples were collected from Zhifu Bay in northern China and analyzed for heavy metals; in addition, their concentrations and pollution status were evaluated. The distributions of Cu and Zn were similar and mainly dominated by fine-grained sediments, whereas the other heavy metal distributions were not very regular. Al was positively correlated with Cu and Zn, and weakly correlated with Pb, As, and Hg. Except for some stations that showed minor enrichment and were unpolluted to moderately polluted by Cr, Cd, and Hg, the overall quality of sediments in the study area was good. Three principal components with eigenvalues >1 were estimated, accounting for 72.06 % of the total variability and representing natural sources, natural and anthropogenic, and anthropogenic sources, respectively.

Heavy metal concentration profiles and pollution assessment in the Jiangsu offshore area, China.

The concentration profiles of various major and trace heavy metals (Cu, Pb, Zn, Cr, Cd, As, Hg, Ni, Li, and Co) were investigated along a 40.1-m-long sediment core in the offshore Jiangsu area of China, to assess their depositional trends and contamination levels. All metals, except Cd, exhibited similar profiles with high average concentrations during the Marine Isotope Stage 2-4 period. The sediment trace-metal concentrations were primarily related to grain size and sediment sources, with almost all heavy metals (except Cd) being positively correlated. Enrichment factors, geoaccumulation indices, and principal component analysis indicated no elemental enrichment or contamination. The high EF and Igeo values of As, Hg, and Li may be related to their background values.

Distribution and pollution assessment of heavy metals in surface sediments along the Weihai coast, China.

In this study, 78 surface sediment samples were collected from the Weihai coastal area and analyzed for heavy metals. Their concentrations and pollution status were evaluated. The distribution of heavy metals was mainly dominated by sediment grain size, and the sediments in the Weihai, Sanggou, and Rushan Bays, which have finer grain sizes, had higher concentrations. The mean geoaccumulation index values for all heavy metals were <0. Expect for Hg, the mean enrichment factor values of the other metals were <1.5, indicating that they are natural sourced. Overall, the environmental quality of the Weihai costal area was relatively good and should be maintained and protected. The heavy metals that had potential impacts on the ecological environment were Cd and Hg, which were mainly distributed west of Weihai Bay and inside Rushan Bay. They are affected by human activities and must be controlled.

The MdmiR156n Regulates Drought Tolerance and Flavonoid Synthesis in Apple Calli and Arabidopsis.

Drought is the major abiotic stress that limits apple productivity and quality. To date, many important and divergent regulatory functions of miR156/SBP genes in plant growth and development have been well understood. However, little is known about the role of apple miR156 in response to abiotic stress. To better understand the functions of MdmiR156 in abiotic stress tolerance, we constructed the overexpression (OE) and short tandem target mimic (STTM) vector of MdmiR156n and performed its functional analysis through the characterization of transgenic apple calli and Arabidopsis thaliana plants. In this study, MdmiR156n overexpression significantly increased the length of primary roots and the number of lateral roots in transgenic Arabidopsis plants under drought stress. In addition, MdmiR156n transgenic Arabidopsis and apple calli had a lower electrolyte leakage rate and less cell membrane damage than WT and STTM156 after drought stress. Further studies showed that MdmiR156n overexpression promoted the accumulation of flavonoids and scavenging of reactive oxygen species (ROS) under drought conditions in transgenic apple calli and A. thaliana plants. Taken together, overexpression MdmiR156n enhances drought tolerance by regulating flavonoid synthesis and ROS signaling cascades in apple calli and A. thaliana.

Geochemical characteristics of heavy metals in surface sediments of the Bohai Strait, China.

The heavy metal (Cr, Cu, Ni, Pb, and Zn) content and particle size of surface sediment samples taken from 123 sites in the Bohai Strait of China were analyzed. All five heavy metals showed a similar distribution in the Bohai Strait, with lower concentrations in the middle and higher concentrations at the sides of the strait. The amount of heavy metals was lowest in the Laotieshan Channel due to the rapid current in this channel. According to our calculated values of the pollution index (Pi), only 1.6 % of the sample sites were polluted by Cr and Cu. The calculated geo-accumulation index (Igeo) values showed that few of the sites were polluted by heavy metals. The level of Pb was controlled by both terrigenous inputs and clay adsorption, while the levels of the other heavy metals (Cr, Cu, Ni, and Zn) were mainly controlled by terrigenous inputs.

Hydrodynamically-driven distribution and remobilization of heavy metals in surface sediments around the coastal area of Shandong Peninsula, China.

Heavy metals (HMs) are considered a major pollutant of the surface sediments of the continental shelf. However, there remains little in-depth research on their fate in the ocean, and particularly on their abundance in sediments and the water column and the underlying drivers. This study examined the concentrations of HMs (Cu, Zn, Cr, Pb, Cd, and As) in surface sediments and suspended particulate matter (SPM) around the coastal area of Shandong Peninsula, China. The division of the sedimentary environment and influencing factors were also analyzed using multivariate statistical analysis Fuzzy c-means (FCM) cluster and Non-Linear Mapping (NLM). The study attempted to understand the distribution and remobilization of HMs in the shallow marginal sea using multi-disciplinary approaches, including satellite remote sensing and numerical simulation. The results showed higher HMs in the surface sediments in Weihai Bay (Zone I) than in the junction of the Chengshantou Cap (Zone III) and north of Wei Bay (Zone II). In addition, the results suggested that Cu, Zn, Cr and Pb originated from natural weathering, with their spatial distributions in the three zones highly regulated by sediment grain size, total nitrogen (TN), and total carbon (TC). In contrast, Cd and As originated from anthropogenic contamination (e.g., industrial discharges and aquaculture) in Zone I. HMs (except As) were influenced by terrigenous total organic carbon (TOC) in Zone III. The results of this study suggest that the difference in sediment re-suspension intensity has an important influence on the distribution of HM concentrations in the north Yellow Sea. This study can act as a reference for understanding the fates and source-sink processes of HMs in offshore sediments. The coupling behaviors and microscopic suspension properties of HMs in surface sediments and SPM require further investigation.

Transcriptome and metabolome analyses reveal phenotype formation differences between russet and non-russet apples.

The apple is an economically important fruit, and fruit russeting is not conducive to its appearance. Although studies have examined fruit russeting, its mechanism remains unclear. Two apple strains of the F1 hybrid population derived from 'Fuji' and 'Golden Delicious' were used in this study. We found that the skin of russet apples was rough and fissured, while that of non-russet apples was smooth and waxy. Chemical staining, LC- and GC-MS showed that both lignin and suberin were increased in russet apple skin. Meanwhile, genes involved in lignin and suberin synthetic pathways were upregulated in russet apple skin. Additionally, we found many differentially expressed genes (DEGs1) involved in hormone biosynthesis and signaling and stress responses in the two apple strains. We found that WRKY13 may influence russeting by regulating lignin synthesis. Our study identified several candidate metabolites and genes, which will provide a good foundation for further research.

Research Progress on Genetic Basis of Fruit Quality Traits in Apple (Malus × domestica).

Identifying the genetic variation characteristics of phenotypic traits is important for fruit tree breeding. During the long-term evolution of fruit trees, gene recombination and natural mutation have resulted in a high degree of heterozygosity. Apple (Malus × domestica Borkh.) shows strong ecological adaptability and is widely cultivated, and is among the most economically important fruit crops worldwide. However, the high level of heterozygosity and large genome of apple, in combination with its perennial life history and long juvenile phase, complicate investigation of the genetic basis of fruit quality traits. With continuing augmentation in the apple genomic resources available, in recent years important progress has been achieved in research on the genetic variation of fruit quality traits. This review focuses on summarizing recent genetic studies on apple fruit quality traits, including appearance, flavor, nutritional, ripening, and storage qualities. In addition, we discuss the mapping of quantitative trait loci, screening of molecular markers, and mining of major genes associated with fruit quality traits. The overall aim of this review is to provide valuable insights into the mechanisms of genetic variation and molecular breeding of important fruit quality traits in apple.

MdMKK9-Mediated the Regulation of Anthocyanin Synthesis in Red-Fleshed Apple in Response to Different Nitrogen Signals.

The mitogen-activated protein kinase (MAPK) signaling cascade is a widely existing signal transduction system in eukaryotes, and plays an important role in the signal transduction processes of plant cells in response to environmental stress. In this study, we screened MdMKK9, a gene in the MAPK family. This gene is directly related to changes in anthocyanin synthesis in the 'Daihong' variety of red-fleshed apple (Malus sieversii f neidzwetzkyana (Dieck) Langenf). MdMKK9 expression was up-regulated in 'Daihong' tissue culture seedlings cultured at low levels of nitrogen. This change in gene expression up-regulated the expression of genes related to anthocyanin synthesis and nitrogen transport, thus promoting anthocyanin synthesis and causing the tissue culture seedlings to appear red in color. To elucidate the function of MdMKK9, we used the CRISPR/Cas9 system to construct a gene editing vector for MdMKK9 and successfully introduced it into the calli of the 'Orin' apple. The MdMKK9 deletion mutants (MUT) calli could not respond to the low level of nitrogen signal, the expression level of anthocyanin synthesis-related genes was down-regulated, and the anthocyanin content was lower than that of the wild type (WT). In contrast, the MdMKK9-overexpressed calli up-regulated the expression level of anthocyanin synthesis-related genes and increased anthocyanin content, and appeared red in conditions of low level of nitrogen or nitrogen deficiency. These results show that MdMKK9 plays a role in the adaptation of red-fleshed apple to low levels of nitrogen by regulating the nitrogen status and anthocyanin accumulation.

Advances in Understanding the Causes, Molecular Mechanism, and Perspectives of Russeting on Tree Fruit.

The external quality of fruit is one of its most important qualities; good external quality attracts consumers easily and increases the value of fruit. Fruit russeting is one of the factors that influences the external quality of fruit and has been studied in most horticultural plants. However, the molecular mechanism of russeting has never been discussed so far. In this review, we summarize the research progress on fruit russeting, including causes, microscopic histomorphology, composition, genetics, and regulation and made a series of elaboration on the current research on fruit russeting. This study aims to provide insights into the mechanisms underlying fruit russeting. It also puts forward ideas for research on fruit russeting, which may provide a reference for future research.

Red-fleshed apple flavonoid extract alleviates CCl4-induced liver injury in mice.

In recent years, the global incidence of liver damage has increased. Despite the many known health benefits of red-fleshed apple flavonoids, their potential liver-protective effects have not yet been investigated. In this study, we analyzed the composition of red-fleshed apple flavonoid extract (RAFE) by high-performance liquid chromatography (HPLC). We then induced liver damage in mice with carbon tetrachloride (CCl4) and performed interventions with RAFE to analyze its effect on liver damage, using bifendate as a positive control. The results showed that catechin was the most abundant flavonoid in 'XJ4' RAFE (49.346 mg/100 g). In liver-injured mice, the liver coefficients converged to normal levels following RAFE intervention. Moreover, RAFE significantly reduced the enzymatic activity levels of glutamic oxaloacetic transaminase (ALT), glutamic alanine transaminase (AST), and alkaline phosphatase (ALP) in mouse serum. Furthermore, RAFE significantly increased the content or enzyme activity level of total glutathione, total antioxidant capacity, and superoxide dismutase, and significantly decreased the content of malondialdehyde in the liver of mice. In parallel, we performed histopathological observations of mouse livers for each group. The results showed that RAFE restored the pathological changes caused by CCl4 around the central hepatic vein in mice and resulted in tightly bound hepatocytes. The recovery effect of RAFE was dose-dependent in the liver tissue. Regarding intestinal microorganisms, we found that RAFE restored the microbial diversity in liver-injured mice, with a similar microbial composition in the RAFE intervention group and normal group. RAFE reduced the ratio of Firmicutes to Bacteroidetes, increased the levels of probiotic bacteria, such as Lactobacillus acidophilus, and Clostridium, and reduced the levels of harmful bacteria, such as Erysipelothrix Rosenbach. Therefore, RAFE ameliorated CCl4-induced liver damage by modulating the abundance and composition of intestinal microorganisms in mice. In conclusion, RAFE alleviated CCl4-induced liver damage in mice, with H-RAFE (5 mg kg-1) significantly improving liver damage in mice but M-RAFE (1 mg kg-1) significantly improving the imbalance of intestinal microorganisms in mice. Our research suggests that RAFE could be employed for the adjuvant treatment and prevention of liver damage, and may have important applications in food and medicine.

Red-Fleshed Apple Anthocyanin Extract Reduces Furan Content in Ground Coffee, Maillard Model System, and Not-from-Concentrate Apple Juice.

Furan is a volatile and carcinogenic heterocyclic chemical compound that occurs in a wide range of thermally processed food. It can be induced during food-preparing processes by high temperatures and UV-C light. In the present study, the degradation of furan content in ground coffee, Maillard model system, and not-from-concentrate (NFC) apple juice by red-fleshed apple anthocyanin extract (RAAE) was studied. The results demonstrated that RAAEs had different degrees of degradation of furan content in coffee powder, and the RAAE from 'XJ3' had the most significant effect, with a reduction rate of up to 20%. Moreover, by adding RAAE to the Maillard model system, we found the amounts of furan were significantly reduced. At the same time, RAAE from 'XJ3' could observably reduce the content of furan in pasteurized NFC juice, with 'Fuli' NFC juice furan content decreasing the most, which was 68%. Taken together, our study demonstrated that the use of RAAE could be a feasible way to reduce furan content in ground coffee, Maillard model system, and NFC apple juice.

The MdHY5-MdWRKY41-MdMYB transcription factor cascade regulates the anthocyanin and proanthocyanidin biosynthesis in red-fleshed apple.

Red-fleshed apple fruits are popular because of their high flavonoid content. Although MdMYB10 and its homologs have been identified as crucial regulators of the fruit coloring process, other transcription factors (TFs) contributing to the differences in flesh coloration have not been fully characterized. In this study, we investigated the regulatory effects of MdWRKY41 on anthocyanin and proanthocyanidin (PA) synthesis in red-fleshed apples. The overexpression of MdWRKY41 in red-fleshed apple calli inhibited anthocyanin and PA accumulation by downregulating the expression of a MYB TF gene (MdMYB12) and specific structural genes (MdLAR, MdUFGT, and MdANR). Furthermore, MdWRKY41 was shown to interact with MdMYB16 to form a complex that can further suppress MdANR and MdUFGT expression. Interestingly, MdWRKY41 was targeted by the photoresponse factor MdHY5 and inhibited its transcription. Overall, our findings provide insights into a novel MdHY5-MdWRKY41-MdMYB regulatory module influencing anthocyanin and PA synthesis in red-fleshed apple fruits.

Ethylene increases the cold tolerance of apple via the MdERF1B-MdCIbHLH1 regulatory module.

Cold stress has always been a major abiotic factor affecting the yield and quality of temperate fruit crops. Ethylene plays a critical regulatory role in the cold stress response, but the underlying molecular mechanisms remain elusive. Here, we revealed that ethylene positively modulates apple responses to cold stress. Treatment with 1-aminocyclopropane-1-carboxylate (an ethylene precursor) and aminoethoxyvinylglycine (an ethylene biosynthesis inhibitor) respectively increased and decreased the cold tolerance of apple seedlings. Consistent with the positive effects of ethylene on cold stress responses, a low-temperature treatment rapidly induced ethylene release and the expression of MdERF1B, which encodes an ethylene signaling activator, in apple seedlings. Overexpression of MdERF1B significantly increased the cold tolerance of apple plant materials (seedlings and calli) and Arabidopsis thaliana seedlings. A quantitative real-time PCR analysis indicated that MdERF1B upregulates the expression of the cold-responsive gene MdCBF1 in apple seedlings. Moreover, MdCIbHLH1, which functions upstream of CBF-dependent pathways, enhanced the binding of MdERF1B to target gene promoters as well as the consequent transcriptional activation. The stability of MdERF1B-MdCIbHLH1 was affected by cold stress and ethylene. Furthermore, MdERF1B interacted with the promoters of two genes critical for ethylene biosynthesis, MdACO1 and MdERF3. The resulting upregulated expression of these genes promoted ethylene production. However, the downregulated MdCIbHLH1 expression in MdERF1B-overexpressing apple calli significantly inhibited ethylene production. These findings imply that MdERF1B-MdCIbHLH1 is a potential regulatory module that integrates the cold and ethylene signaling pathways in apple.

MdMYB114 regulates anthocyanin biosynthesis and functions downstream of MdbZIP4-like in apple fruit.

Anthocyanins, a major class of compounds derived from the flavonoid pathway, are important pigments of apple fruit. They can also prevent certain diseases and are beneficial to human health. Fruit pigmentation is a key quality trait that influences consumer preference; therefore, it is of great importance to investigate its regulatory mechanism. Here, we identified a MYB transcription factor (TF), MdMYB114, whose transcript level increased in the skin of the deep red apple fruit. It was determined to belong to the R2R3-MYB TF family and was localized in the nucleus. MdMYB114 overexpression led to anthocyanin accumulation in apple calli. MdMYB114 was not able to form an MBW complex but could enhance anthocyanin biosynthesis and transport by directly binding to the promoters of MdANS, MdUFGT, and MdGST to promote their expression. In addition, multiple assays revealed that MdbZIP4-like, a basic leucine-zipper TF, could directly bind to the MdMYB114 promoter to enhance its expression. Taken collectively, our results provide evidence that MdMYB114 is a positive regulator of anthocyanin biosynthesis and transport and it functions downstream of MdbZIP4-like in apple fruit.

HEAT SHOCK FACTOR A8a Modulates Flavonoid Synthesis and Drought Tolerance.

Drought is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus domestica). Heat shock factors (HSFs) have well-documented functions in stress responses, but their roles in flavonoid synthesis and the flavonoid-mediated drought response mechanism remain elusive. In this study, we demonstrated that a drought-responsive HSF, designated MdHSFA8a, promotes the accumulation of flavonoids, scavenging of reactive oxygen species, and plant survival under drought conditions. A chaperone, HEAT SHOCK PROTEIN90 (HSP90), interacted with MdHSFA8a to inhibit its binding activity and transcriptional activation. However, under drought stress, the MdHSP90-MdHSFA8a complex dissociated and the released MdHSFA8a further interacted with the APETALA2/ETHYLENE RESPONSIVE FACTOR family transcription factor RELATED TO AP2.12 to activate downstream gene activity. In addition, we demonstrated that MdHSFA8a participates in abscisic acid-induced stomatal closure and promotes the expression of abscisic acid signaling-related genes. Collectively, these findings provide insight into the mechanism by which stress-inducible MdHSFA8a modulates flavonoid synthesis to regulate drought tolerance.

MdMYB6 regulates anthocyanin formation in apple both through direct inhibition of the biosynthesis pathway and through substrate removal.

Anthocyanin biosynthesis and sugar metabolism are important processes during plant growth, but the molecular interactions underlying these pathways are still unclear. In this work, we analyzed the anthocyanin and soluble sugar contents, as well as the transcript levels of transcription factors that are known to be related to the biosynthesis of anthocyanin in 'Hongcui 1' apple flesh during fruit development. Overexpression of MdMYB6 in red-fleshed calli was found to reduce anthocyanin content and result in downregulated expression of the MdANS and MdGSTF12 proteins. Yeast one-hybrid and electrophoretic mobility shift analyses showed that MdMYB6 could directly bind to the promoters of MdANS and MdGSTF12, indicating that MdMYB6 could inhibit anthocyanin biosynthesis by regulating MdANS and MdGSTF12. Overexpression of MdTMT1 in the Arabidopsis tmt1 mutant restored the glucose and fructose contents to the wild-type levels, while overexpression of MdTMT1 in red-fleshed calli increased the contents of glucose and fructose but reduced the contents of UDP-glucose, UDP-galactose, and anthocyanin. Using a GUS reporter system, yeast one-hybrid, chromatin immunoprecipitation-PCR and electrophoretic mobility shift analyses, we found that MdMYB6 could bind to the promoter of MdTMT1, resulting in increased promoter activity. Overexpression of MdMYB6 in calli overexpressing MdTMT1 increased the expression of MdTMT1, which led to reduced contents of UDP-glucose and UDP-galactose and decreased anthocyanin content compared to those of the calli that overexpressed MdTMT1. This finding suggested that MdMYB6 could also inhibit anthocyanin biosynthesis by regulating MdTMT1 to decrease the contents of UDP-glucose and UDP-galactose. Taken together, these results showed that MdMYB6 and MdTMT1 play key roles in both anthocyanin biosynthesis and sugar transport.