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.

Profiling and optimized extraction of bioactive polyphenolic compounds from young, red-fleshed apple using eco-friendly deep eutectic solvents.

Red-fleshed apple cultivars with an enhanced content of polyphenolic compounds have attracted increasing interest due to their promising health benefits. Here, we have analysed the polyphenolic content of young, red-fleshed apples (RFA) and optimised extraction conditions of phenolics by utilising natural deep eutectic solvents (NDES). We also compare the antioxidant, neuroprotective and antimicrobial activities of NDES- and methanol-extracted phenolics from young RFA. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was used for phenolics identification and quantification. Besides young RFA, ripe red-fleshed, young and ripe white-fleshed apples were analysed, revealing that young RFA possess the highest phenolic content (2078.4 ± 4.0 mg gallic acid equivalent/100 g), and that ripe white-fleshed apples contain the least amount of phenolics (545.0 ± 32.0 mg gallic acid equivalent/100 g). The NDES choline chloride-glycerol containing 40 % w/w H2O gave similar yields at 40 °C as methanol. In addition, the polyphenolics profile, and bioactivities of the NDES extract from young RFA were comparable that of methanol extracts. Altogether, our data show that NDES extracts of young RFA are a promising source of bioactive polyphenolics with potential applications in diverse sectors, e.g., for functional food production, smart material engineering and natural therapies.

Unraveling the Mechanism of Cork Spot-like Physiological Disorders in 'Kurenainoyume' Apples Based on Occurrence Location.

Cork spot-like physiological disorder (CSPD) is a newly identified issue in 'Kurenainoyume' apples, yet its mechanism remains unclear. To investigate CSPD, we conducted morphological observations on 'Kurenainoyume' apples with and without pre-harvest fruit-bagging treatment using light-impermeable paper bags. Non-bagged fruit developed CSPD in mid-August, while no CSPD symptoms were observed in bagged fruit. The bagging treatment significantly reduced the proportion of opened lenticels, with only 17.9% in bagged fruit compared to 52.0% in non-bagged fruits. In non-bagged fruit, CSPD spots tended to increase from the lenticels, growing in size during fruit development. The cuticular thickness and cross-sectional area of fresh cells in CSPD spots were approximately 16 µm and 1600 µm², respectively. Healthy non-bagged fruit reached these values around 100 to 115 days after full bloom from mid- to late August. Microscopic and computerized tomography scanning observations revealed that many CSPD spots developed at the tips of vascular bundles. Therefore, CSPD initiation between opened lenticels and vascular bundle tips may be influenced by water stress, which is potentially caused by water loss, leading to cell death and the formation of CSPD spots.

Red-Fleshed Apple Flavonoids Extract Alleviates Male Reproductive Injury Caused by Busulfan in Mice.

In this research, we analyzed the protective effects of red-fleshed apple flavonoid extracts (RAFEs) on male reproductive injury induced by busulfan, using both in vitro and in vivo models. In the cell-based experiments, RAFEs significantly improved cell viability and proliferation rates compared to control groups. Similarly, in vivo testing with male mice showed that RAFEs and whole apple flavonoid extracts (WAFEs) enhanced various biochemical and liver function-related indicators in the testes; however, RAFEs demonstrated superior efficacy in mitigating testicular damage. Through immunohistochemistry, qRT-PCR, and Western blotting, we found that RAFEs notably enhanced the expression of spermatogenesis-related genes. Moreover, RAFEs increased the expression of oxidative stress- and apoptosis-related genes, thereby effectively reducing oxidative damage in the testes. These findings highlight the potential of RAFEs as natural agents for the prevention and treatment of male reproductive injury, paving the way for future research and potential therapeutic applications.

MdMYB305-MdbHLH33-MdMYB10 regulates sugar and anthocyanin balance in red-fleshed apple fruits.

Sugar and anthocyanin are important indicators of fruit quality, and understanding the mechanism underlying their accumulation is essential for breeding high-quality fruit. We identified an R2R3-MYB transcription factor MdMYB305 in the red-fleshed apple progeny, which was positively correlated with fruit sugar content but negatively correlated with anthocyanin content. Transient injection, stable expression [overexpressing and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)], and heterologous transformation of tomato confirmed that MdMYB305 promotes the accumulation of sugar and inhibits the synthesis of anthocyanin. A series of molecular experiments (such as electrophoretic mobility shift and luciferase assays) confirmed that MdMYB305 combines with sugar-related genes (MdCWI1/MdVGT3/MdTMT2) and anthocyanin-related genes (MdF3H/MdDFR/MdUFGT), promoting and inhibiting their activities, and finally regulating the sugar and anthocyanin content of fruits. In addition, the study also found that MdMYB305 competes with MdMYB10 for the MdbHLH33 binding site to balance sugar and anthocyanin accumulation in the fruits, which provides a reference value for exploring more functions of the MYB-bHLH-MYB complex and the balance relationship between sugar and anthocyanin in the future.

Mdm-miR858 targets MdMYB9 and MdMYBPA1 to participate anthocyanin biosynthesis in red-fleshed apple.

Anthocyanins are important secondary metabolites in plants. They are important for human health because of their antioxidant activities and because their dietary intake reduces the incidence of cardiovascular and cerebrovascular diseases and tumors. The biosynthesis of anthocyanins and its regulation in fruits and vegetables is a global research hotspot. Compared with cultivated apples, the red-fleshed apple is a relatively new and popular commodity in the market. Previous studies on red-fleshed apples have focused on the basis for the high anthocyanin content and the transcriptional regulation of anthocyanin synthesis. In the present study, we focused on the mechanism of microRNA-mediated post-transcriptional regulation of anthocyanin synthesis in red-fleshed apples. We identified a microRNA (miRNA), designated mdm-miR858, that is specifically expressed in the flesh of apple fruit. The expression level of miR858 was significantly lower in red-fleshed apples than in white-fleshed apples. The overexpression of mdm-miR858 significantly inhibited anthocyanin accumulation, whereas the silencing of mdm-miR858 promoted anthocyanin synthesis in STTM858 transgenic apple calli. Further analyses showed that mdm-miR858 targets the transcription factor genes MdMYB9 and MdMYBPA1 to participate anthocyanin accumulation in apple. Our results also show that MdHY5, a transcription factor in the light signaling pathway, can bind to the promoter of mdm-miR858 to inhibit its transcription, thereby regulating anthocyanin synthesis. Based on our results, we describe a novel HY5-miR858-MYB loop involved in the modulation of anthocyanin biosynthesis. These findings provide new information about how plant miRNAs regulate anthocyanin anabolism and provide a basis for breeding new anthocyanin-rich, red-fleshed apple varieties.

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.

MdJa2 Participates in the Brassinosteroid Signaling Pathway to Regulate the Synthesis of Anthocyanin and Proanthocyanidin in Red-Fleshed Apple.

Anthocyanin and proanthocyanidin play important roles in plant secondary metabolism. Although previous studies identified many transcription factors involved in anthocyanin and proanthocyanidin synthesis, the effects of MADS-box transcription factors are unclear in apple. Brassinosteroids (BRs) are steroid hormones that affect plant flavonoid biosynthesis, but the underlying regulatory mechanism is not yet well established. In this study, we identified a MADS-box transcription factor, MdJa2, which contained a highly conserved MADS-box domain and belonged to the STMADS11 subfamily. Additionally, MdJa2 was responsive to BR signal, and the overexpression of MdJa2 inhibited the synthesis of anthocyanin and proanthocyanidin. The silencing of MdJa2 in "Orin" calli promoted anthocyanin and proanthocyanidin accumulations. Moreover, MdJa2 interacted with MdBZR1. MdJa2 was revealed to independently regulate anthocyanin and proanthocyanidin synthesis pathways. The MdJa2-MdBZR1 complex enhanced the binding of MdJa2 to the promoters of downstream target genes. Our research provides new insights into how MADS-box transcription factors in the BR signaling pathway control the accumulations of anthocyanin and proanthocyanidin in red-fleshed apple.

Phenol metabolic fingerprint and selection of intake biomarkers after acute and sustained consumption of red-fleshed apple versus common apple in humans. The AppleCOR study.

The present study aimed to evaluate the metabolism and bioavailability of anthocyanins (ACN) and other phenolics from red-fleshed apple (RFA) and to define the intake biomarkers compared to common white-fleshed apple (WFA). Acute and sustained (6-week) interventions were combined in a randomized, controlled and parallel study with 121 hypercholesterolemic subjects. Another arm consuming ACN-rich infusion from aronia fruit (ARO) provided matched content and profile of ACN. Plasma, urine and faeces samples were analyzed using UPLC-MS/MS. Results showed higher bioavailability of ACN after ARO compared to RFA, showing a clear apple matrix effect. The dihydrochalcone phloretin-2'-O-glucuronide was the most discriminant intake biomarker of both apples. The urinary peonidin-3-O-galactoside was a good biomarker after both ARO and RFA intakes, whereas peonidin-O-arabinoside was reported to be specific from ARO. The elucidation of the phenolic metabolism and the selection of intake biomarkers is a promising approach to relate phenolic compounds and human health.

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.

Evolution of typical aromas and phenolic compounds of a red-fleshed apple throughout different fruit developmental periods in Xinjiang, China.

Red-fleshed apple is attracting more and more attentions from both consumers and researchers thanks to its pleasant appearance and flavor. Accumulation and development of aromas of apple fruit are important factors to understand the fruit quality of a new cultivated variety. However, knowledge on the key aroma components development and accumulation of red-fleshed apple (Malus sieversii f. neidzwetzkyana(Dieck) Langenf) in Xinjiang through the fruit developmental periods is insufficient. In this paper, free and bound form aromas of a red-fleshed apple since young fruit period through expansion period and conversion period to ripen period were comparatively studied. We classified aroma substances according to the compound category (esters, aldehydes, alcohols, terpenes, and aromatics), and to the odor types. The concentration of bound form aroma compounds were higher than that of free form throughout the 4 periods and showed different evolution trends. Among the 4 periods, the bound aroma was the most in the expansion period; and terpenoids showed the greatest potential to release free form aromas. Total aromas were found the lowest concentration in expansion period and the highest concentration in ripen period. The observations in this study will provide theoretical support for the research of typical aroma formation regularity of red-fleshed apple, and guide for the aroma enhancement regulation.

Differential effects of phenolic extracts from red-fleshed apple peels and flesh induced G1 cell cycle arrest and apoptosis in human breast cancer MDA-MB-231 cells.

Red-fleshedapples are preferredbecause of their high content of phenolics and antioxidants in peel and pulp. Herein, we evaluated the mechanisms of apple peel polyphenolic extracts (APP) and apple flesh polyphenolic extracts (AFP) from the new red-fleshed apple in inhibiting cell proliferation and inducing apoptosis on human breast cancer MDA-MB-231 cells. The antiproliferative activities were determined by the CCK8 assay. The expression of proteins was determined using Western blot. We found that the content of polyphenols and flavonoids in APP was significantly higher than that in AFP, and 14 main phenolic compounds in APP and AFP were quantified using UPLC-MS/MS techniques. Besides, the significant inhibition effects of APP and AFP were achieved through Akt pathway by inducing apoptosis (significantly upregulating reactive oxygen species [ROS] levels, and downregulating expression of pAkt, pBad, Bcl-2, promoting Cytochrome c release, activating Cle-Caspase 9, and inducing expressions of Cle-Caspase 3 and Cle-PARP), and inducing G0/G1 cell cycle arrest (increased expressions of p-p53 and p21 and decreased expressions of PCNA and Cyclin D1). And the inhibition effect of APP was stronger than that of AFP. These results suggest that AFP and APP may be excellent sources of natural chemicals for treating triple-negative breast cancer MDA-MB-231 cells. PRACTICAL APPLICATION: The effects of antiproliferation of phenolic extracts from red-fleshed apple peels and flesh on human breast cancer MDA-MB-231 cells were evaluated. The data may clarify the functional parts of red-fleshed apple and provide some basis for scientific researchers and consumers to recognize and exploit red-fleshed apple.

Red-Fleshed Apple Anthocyanin Extracts Attenuate Male Reproductive System Dysfunction Caused by Busulfan in Mice.

In this research, we analyzed the effect of an intragastrical oral administration of red-fleshed apple anthocyanin extract (RAAE) on busulfan-treated mice. First, we showed that the most abundant component in RAAE was cyanidin 3-O-galactoside. To determine the effect of the RAAE, the mice were divided into control and four other different concentrations of RAAE feeding treatment groups (BA0, no RAAE; BA.1, 0.1 mg/kg; BA1, 1 mg/kg; and BA5, 5 mg/kg) following busulfan injection. We observed that RAAE treatments displayed ameliorative effects on male reproductive system dysfunction caused by busulfan, such as recovering the irregular arrangements of seminiferous tubules, increasing the number of spermatogonia and spermatocytes, improving sperm concentration by 3-fold in BA.1, and improving sperm motility by 2-fold in BA1. The liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis showed significant up- or downregulation of certain metabolites, such as lysophosphatidylcholine (LysoPC), L-arginine, glycine, anandamide, and L-carnitine, which could contribute to the positive effects of RAAE, especially in PBA1 (plasma of BA1) and PBA5 (plasma of BA5). Taken together, the results indicate that 1 mg/kg of RAAE is a suitable concentration for rescuing spermatogenesis in mice. The research suggests that RAAE could be a potential nutraceutical for protecting spermatogenesis after busulfan therapy in cancer.

Metabolic Fate and Cardiometabolic Effects of Phenolic Compounds from Red-Fleshed Apple in Hypercholesterolemic Rats: A Comparative Study with Common White-Fleshed Apple. The AppleCOR Study.

The present study aims to investigate the metabolic fate and the cardiometabolic effects of phenolic compounds provided by a red-fleshed apple variety biofortified in anthocyanins (ACN). Wistar rats are fed with high-fat diet (HFD) to induce hypercholesterolemia and supplemented with red-fleshed apple (HFD+R), white-fleshed apple (HFD+W), or an ACN-rich infusion from aronia fruit (HFD+A) providing matched content and profile of ACN. Plasma biochemical parameters, histological analysis, and phenol biological metabolites are determined. Plasma, urine, and feces show a significant increase of ACN metabolites after HFD+R and HFD+A, while flavan-3-ols are significantly increased after HFD+W and dihydrochalcones derivatives increased after both apples supplementation. A cardioprotective effect is observed after both apples and aronia infusion supplementation in the reduction of aortic thickness. The kidney function is improved after all supplementations and a decrease in insulin plasma concentration after both apples supplementation (HFD+R and HFD+W) is also observed. The findings support that ACN without apple matrix can induce cardioprotective effects. ACN or flavan-3-ols, together with dihydrochalcones, compose a phenolic phytocomplex in red- and white-fleshed apples, respectively, which can act synergistically in the attenuation of cardiovascular outcomes in hypercholesterolemic rats.

Combined Analysis of Gut Microbiota and Plasma Metabolites Reveals the Effect of Red-Fleshed Apple Anthocyanin Extract on Dysfunction of Mice Reproductive System Induced by Busulfan.

Busulfan is currently an indispensable anti-cancer drug, but the side effects on male reproductive system are so serious. Meanwhile, red-fleshed apples are natural products with high anthocyanin content. In this research, we analyzed the effect of red-fleshed apple anthocyanin extract (RAAE) on busulfan-treated mice. Compared with the busulfan group, main plasma biochemical indicators were significantly improved after RAAE treatment. Compared with BA0 (busulfan without RAAE) group, total antioxidant capacity(T-AOC) and the activity of superoxide dismutase (SOD) and glutathione catalase (GSH-Px) in RAAE treatment groups were obviously increased, while the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly decreased. Malondialdehyde (MDA) was significantly decreased in the RAAE groups. In addition, we found RAAE alleviated busulfan-disrupted spermatogenesis through improving genes expression which are important for spermatogenesis, such as DDX4, PGK2, and TP1. Furthermore, we found that RAAE increased beneficial bacteria Akkermansia and Lactobacillaceae, and significantly depleted harmful bacteria Erysipelotrichia. The correlation studies indicated that RAAE ameliorated busulfan-induced rise in LysoPC levels through regulating gut microbial community and their associated metabolites. In conclusion, this study extends our understanding of the alleviated effect of RAAE on busulfan-induced male reproductive dysfunction through regulating the relationships between gut microbiota and metabolites.

Effects of Different Drying Methods on the Retention of Bioactive Compounds, On-Line Antioxidant Capacity and Color of the Novel Snack from Red-Fleshed Apples.

The aim of this study was to determine the effect of different drying methods: convective (at 50, 60, 70 °C), vacuum-microwave (at 120, 240, 360, 480 W and 360 W with reduction to 120 W) and hybrid (convective pre-drying at 50, 60, 70 °C followed by vacuum-microwave drying at 120 W) on the quality parameters of novel red-fleshed apple fruit snacks (RFAs), such as phenolics, on-line antioxidant capacity, water activity and color. Drying kinetics, including a temperature profile of dried material, and modified Page model were determined. Freeze-drying was used as a control method. The highest content of bioactive compounds in the samples was retained following freeze-drying, then hybrid, vacuum-microwave and finally convection drying. The antioxidant capacity measured by on-line 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), identified anthocyanins, flavan-3-ols and phenolic acid as the main compounds responsible for this activity. Unfavorable changes in color, formation of hydroxymethylfurfural (HMF) and degradation of polyphenolics were noted along with increasing drying temperature and magnetron power. The red-fleshed apple snacks are a promising high-quality dehydrated food product belonging to functional foods category.

Antioxidant and hepatoprotective effects against acute CCl4 -induced liver damage in mice from red-fleshed apple flesh flavonoid extract.

Apple (Malus domestica Borkh.) is an important fruit tree species worldwide. Apple fruits are favored by consumers because of their antioxidative, anti-inflammatory, and antitumor effects as well as their protective effects against cardiovascular diseases and other health benefits. There is considerable interest in red-fleshed apple fruits among breeders because of their high flavonoid and anthocyanin contents. However, the flavonoids extracted from red-fleshed apple fruits must still be functionally characterized, especially regarding their protective effects against certain pathologies. In this study, the flavonoid components and contents in the extracts prepared from red-fleshed apple cultivar "Meihong" were determined. Additionally, the in vitro antioxidant activities and protective effects of the extracts against CCl4 -induced acute liver injury were investigated. The red-fleshed apple flesh flavonoid extract (RAFF) exhibited strong in vitro antioxidant activities. Compared with the model control mice treated with CCl4 , the mice pretreated with high (800 mg/kg·bw), middle (400 mg/kg·bw), and low (200 mg/kg·bw) RAFF doses had significantly lower CCl4 -induced serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities. Moreover, the RAFF pretreatment also significantly decreased the liver malondialdehyde activity and prevented the CCl4 -induced decrease in liver superoxide dismutase, glutathione peroxidase, catalase, and reduced glutathione levels. Furthermore, a histopathological examination revealed that RAFF inhibited the inflammatory cell infiltration and cell boundary loss caused by CCl4 in the liver. Thus, RAFF is a natural antioxidant with significant antioxidative activities and liver protective effects. The results of this study may be relevant for enhancing the application of the red-fleshed apple fruit extract as a food additive. PRACTICAL APPLICATION: We took the self-selected red-fleshed apple cultivar "Meihong" as the unique research material, and the active ingredients of its flavonoid extract, in vitro antioxidant activity and hepatoprotective effect were analyzed. It is of great significance to promote the development of the red-fleshed apple industry, and also provides an important reference for the development of natural antioxidants.

Analysis of Antioxidant Activity and Flavonoids Metabolites in Peel and Flesh of Red-Fleshed Apple Varieties.

In this research, we compared the phenotypical characters, total anthocyanins content, total phenols content, and antioxidant activity of red-fleshed apple cultivars 'XJ4', 'QN-5', 'DH' and 'HX1' at three fruit developmental stages. A further flavonoids metabolites study was conducted in 'XJ4' and 'DH'. We found broader variation of total anthocyanins content in the peel of the four cultivars, which might result in larger differences of free radicals scavenging rate. The most significant difference in fruit phenotype, anthocyanins content, and DPPH scavenging rate was observed between 'XJ4' and 'DH' at mature stage. Therefore, the flavonoids metabolites of 'XJ4' and 'DH' at mature stage were compared to unveil the details of anthocyanins compounds. The unique compounds pelargonidin 3-O-ß-d-glucoside and cyanidin-3-O-malonylhexoside were detected only in peel and flesh of 'XJ4' but not in 'DH', which might contribute to the purple peel and dark-red flesh color of 'XJ4'. Significantly decreased upstream metabolites in the early biosynthetic genes regulated domain were found only in 'XJ4' peel but not in the flesh. This might explain why the anthocyanins content in 'XJ4' peel was decreased largely at the mature stage. Taken together, our findings will give some insight into the metabolites study in flavonoid biosynthetic pathway of red-fleshed apple.

A novel NAC transcription factor, MdNAC42, regulates anthocyanin accumulation in red-fleshed apple by interacting with MdMYB10.

Anthocyanin pigmentation is an important consumption trait of apple (Malus domestica Borkh.). In this study, we focused on the identification of NAC (NAM, ATAF1/2 and CUC2) proteins involved in the regulation of anthocyanin accumulation in apple flesh. A group of MdNACs was selected for comparison of expression patterns between the white-fleshed cultivar 'Granny Smith' and red-fleshed 'Redlove'. Among them, MdNAC42 was screened, which exhibited a higher expression level in red-fleshed than in white-fleshed fruit, and has a positive correlation with anthocyanin content as fruits ripened. Moreover, overexpression of MdNAC42 in apple calli resulted in the up-regulation of flavonoid pathway genes, including MdCHS, MdCHI, MdF3H, MdDFR, MdANS and MdUFGT, thereby increasing the accumulation of anthocyanins, which confirmed the roles of MdNAC42 in anthocyanin biosynthesis. Notably, MdNAC42 was demonstrated to have an obvious interaction with MdMYB10 either in vitro or in vivo by yeast two-hybrid combined with bimolecular fluorescence complementation, further suggesting that MdNAC42 is an important part of the regulatory network controlling the anthocyanin pigmentation of red-fleshed apples. To the best of our knowledge, this is the first report identifying the MdNAC gene as related to anthocyanin accumulation in red-fleshed apples. This study provides valuable information for improving the regulatory model of anthocyanin biosynthesis in apple fruit.