Fruit russeting adversely affects apple appearance and increases loss of moisture after post-harvest, thereby affecting shelf life, storage, and transport. Therefore, it is important to explore appropriate regulation strategies to alleviate apple fruit russeting. In this study, the russet-alleviating effects of prohexadione calcium (P-Ca), GA4 + 7 and Guoxiujing were evaluated in ‘Hongdan’ apple and the results demonstrated that 250 mg/L P Ca exhibited the optimal anti-russeting capacity, with an average russet control rate of 60.7%. P-Ca maintained cuticle integrity, promoted wax accumulation and suppressed suberin deposition by modulating relevant biosynthetic genes. Meanwhile, P-Ca improved fruit crispness, soluble sugar and anthocyanin level in fruit peels. Additionally, the AP2/ERF transcription factor MdSHN2-like was identified as a positive regulator of wax biosynthesis. This study revealed the mechanism by which P-Ca inhibits the russeting formation through promoting wax accumulation in ‘Hongdan’ apple, and provided a theoretical basis for the prevention of fruit russeting in horticultural crops.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease globally, yet effective therapeutic options remain limited. Red-fleshed apples are rich in dietary flavonoids, but their chemical basis and therapeutic potential for MASLD have not been systematically explored. This study integrated LC-MS/MS metabolomics with a high-fat diet (HFD)-induced MASLD mouse model to evaluate the therapeutic effects and mechanisms of 'XJ4' red-fleshed apple flavonoid extracts (RAFEs). Metabolomics identified 120 types of flavonoids in white-fleshed apple 'FJ' and red-fleshed apple 'XJ4', and 57 differentially accumulated metabolites have been detected in both, among which 39 flavonoids significantly accumulated higher in 'XJ4'. Compared with 'FJ', 'XJ4' was predominantly enriched in O-glycosylated flavonols, including isorhamnetin 3-O-glucoside, cacticin, tamarixin, reynoutrin, and guaijaverin. Male ICR mice were randomly divided into nine groups (n = 10): three groups, normal control, HFD model control, and positive control, receiving simvastatin, 10 mg kg-1, and six groups receiving RAFEs or white-fleshed apple flavonoid extracts (WAFEs) at low, medium, or high doses (1, 3 and 5 mg kg-1). Hepatic parameters were assessed by histopathological analysis, biochemical assays, RT-qPCR, immunofluorescence, and western blot analysis; the gut microbiota composition was analysed by 16S rRNA gene sequencing. Medium-dose RAFEs (3 mg kg-1) conferred optimal efficacy, significantly reducing body weight gain, liver coefficient, and plasma ALT, AST, and ALP levels while restoring the hepatic histological architecture. Mechanistically, RAFEs suppressed pro-inflammatory mediators (IL-6, IL-1β, NF-κB, IRF6 and TLR4) and the oxidative stress marker CYP2E1, while enhancing antioxidant capacity (SOD, CAT and T-AOC). RAFEs also reduced hepatic TG, TC, and LDL-C, increased HDL-C, and modulated lipid metabolism via AMPK and PPAR-α upregulation with α-SMA suppression. Furthermore, RAFEs restored gut microbiota diversity, enriched beneficial taxa (Lactobacillus johnsonii, Bifidobacterium pseudolongum and Bacteroides acidifaciens), and suppressed pathogenic Desulfovibrio fairfieldensis. RAFEs consistently outperformed WAFEs, attributable to XJ4's unique isorhamnetin-dominated flavonol glycoside profile. These findings support red-fleshed apple flavonoids as promising natural agents for MASLD treatment.

Glomerella leaf spot (GLS) is a devastating disease of apple. The defense mechanisms of apple plants against GLS remain incompletely understood. In the present study, we report a GLS resistance regulatory module in apple involving MdLBD41, MdbHLH144, MdTPL1, and the target genes MdRBOHD1/2, which are responsible for reactive oxygen species (ROS) generation during infection by Colletotrichum fructicola, a causal agent of GLS. Compared with the resistant cultivar, the susceptible cultivar exhibited a higher expression level of MdLBD41 in response to C. fructicola infection, suggesting the involvement of MdLBD41 in the defense responses of apple plants against GLS. This differential expression of MdLBD41 was associated with distinct levels of histone acetylation in the MdLBD41 promoter region between susceptible and resistant cultivars. Functioning as a transcriptional repressor, MdLBD41 suppressed MdRBOHD1/2 expression and reduced ROS generation by recruiting the TOPLESS corepressor MdTPL1. This repression could be relieved by MdbHLH144, which interacted with MdLBD41 and inhibited its binding to the MdRBOHD1/2 promoters. Thus, we identified a previously uncharacterized regulatory mechanism governing MdRBOHD1/2 expression and ROS generation during C. fructicola infection. This mechanism represents a finely tuned regulatory strategy that balances effective disease resistance with protection against cellular damage in apple plants.

Fruit russeting is a physiological disorder that severely impairs external appearance, reduces commercial value and impacts the shelf-life of apple fruit. Although several transcription factors have been identified in russeting formation, the underlying regulatory mechanisms remain incompletely understood. Through the study of the fruits of russet and non-russet strains, we found that the formation of fruit russeting was related to the lignin content and then we characterized the role of MdWRKY13 in regulating lignin biosynthesis during apple fruit russeting formation. Expression analysis revealed that MdWRKY13 was highly expressed during russeting formation, and subcellular localization assays confirmed its nuclear localization. Transient transformation experiments revealed that MdWRKY13 positively regulates lignin accumulation in apple peel, while stable overexpression in ‘GL-3’ apple seedlings promoted lignin accumulation in stems. Y1H, ChIP-PCR and LUC assays demonstrated that MdWRKY13 directly bound to the promoters of the lignin biosynthetic genes C4H and F5H, activating their transcription. Our findings reveal that MdWRKY13 contributes to russeting formation by promoting lignin biosynthesis through direct transcriptional activation of C4H and F5H, providing new insights into the molecular mechanisms underlying apple fruit russeting and identifying potential targets for fruit quality and storage improvement.

Cuticular wax plays a crucial role in reducing water loss, enhancing pathogen resistance, and improving the surface appearance of apple fruit. However, limited research has focused on wax biosynthesis during fruit development in apple. In this study, we investigated the wax biosynthesis of four apple cultivars 'Fumei', 'Fuxing', 'Fuli', and 'Fuji'. Among them, 'Fumei' apple exhibited significantly higher wax accumulation than the other three cultivars, reaching up to 4,604,997.01 μg/dm2. Scanning electron microscopy (SEM) revealed distinct wax formation patterns among the cultivars, characterized by the development of wax structures on the fruit surface. Notably, in 'Fumei', the cuticular wax layer began to thicken at 150 days after full bloom (DAFB), reaching a significantly greater thickness than that observed in the other cultivars. Gas chromatography-mass spectrometry (GC-MS) analysis revealed a substantial increase in wax content in 'Fumei' during the later stages of fruit development, with alkanes comprising 50.39 % of the total wax. Furthermore, we identified a MYB transcription factor, MdMYB94, which positively regulates wax accumulation in apple fruit. Our findings provide novel insights into the physiological and molecular characteristics of 'Fumei' as a high-wax germplasm, offering valuable theoretical and genetic resources for improving apple fruit appearance and enhancing stress resistance through breeding programs.

‘Fumei’ apple is characterized by high anthocyanin content and thick wax layer. Long non-coding RNAs (lncRNAs) play essential roles in the growth and development of various plants via regulation of gene expression. This study explored the potential mechanism underlying anthocyanin accumulation and cuticular wax formation during the development of ‘Fumei’ apple fruit. The results demonstrated that anthocyanin accumulation correlates with fruit coloration, while wax content drives wax layer formation. A total of 6039 and 3410 differentially expressed genes (DEGs), as well as 230 and 131 differentially expressed lncRNAs (DELs) were identified in the M1/M2 and M2/M3 pairs, respectively, by using RNA-seq. In the M1/M2 pair, the DEGs were mainly enriched in the ‘photosynthesis’ and ‘flavonoid biosynthesis’ pathways; in the M2/M3 pair, the DEGs were significantly enriched in the ‘photosynthesis’ and ‘cutin, suberine and wax biosynthesis’ pathways. Furthermore, the structural and regulatory genes involved in anthocyanin and cuticular wax biosynthesis were investigated, and the potential lncRNAs and genes that may control the anthocyanin and cuticular wax biosynthesis were identified. This study provides candidate lncRNAs and potential regulatory genes associated with both the regulation of anthocyanins and wax during apple development.

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 R-6 promoter, but R-6: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 R-6 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 R-6 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.

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.

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.

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.

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.

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.

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.

This review summarized the research progress of plant epigenetics and fruit tree bud sports selection, and analyzed the classic cases of bud sports selection promoting the high-quality and efficient development of the word's apple and citrus industries. The main results were summarized as follows: (1) Epigenetics caused by DNA methylation and histone modification, etc., was widely involved in various plant growth and development processes and adversity stress responses; (2) The selection of fruit tree bud sports was the best among the best. It had six characteristics, such as high efficiency, reproducibility, stability, diversity and pleiotropy, epigenetic characteristics, and practical effects. At present, more than 600 varieties of fruit tree bud sports have been selected, such as apples and citrus; (3) In response to the problems of the four varieties of Fuji, Delicious, Navel Oranges and Satsuma Mandarin, a series of new varieties have been bred using continuous multi-generation bud sports selection technology, forming a huge variety group and promoting the high-quality and efficient development of the industry. Therefore, two aspects should be further studied in the future: one is to further strengthen the mechanism of fruit tree bud sports and to promote the innovation of breeding technology; The second is to further enhance the awareness of the innovation of fruit tree bud sports selection and to further strengthen the understanding of the importance of the fruit tree industry, and the technical route of combining Mendelian inheritance and epigenetic inheritance should be adopt, combining conventional and molecular technology as well as hybrid breeding and bud sports selection, so as to increase the research efforts of fruit tree bud sports selection and new varieties breeding, and provide varieties support for the high-quality development of the fruit tree industry.

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.

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.

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 MdHY5MdWRKY41-MdMYB regulatory module influencing anthocyanin and PA synthesis in red-fleshed apple fruits.

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.

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.