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The replant disease refers to abnormal crop growth caused by continuous cultivation of the same or related species on the same soil, and often occurring in crops and orchards. Recently, replant dis...

Soil salinization is one of the most important abiotic stresses adversely affecting plant growth and yield. In this study, we cloned and characterized a putative salt tolerance gene encoding a bHLH (basic helix-loop-helix) transcription factor, MdbHLH106L (MdbHLH106-like), in the genome of apple (Malus domestica Borkh.). The expression level and promoter activity of MdbHLH106L are increased under salt stress. Overexpressing MdbHLH106L in ‘Orin’ calli can promote the expression level of MdNHX1 (Na+/H+ exchanger 1). Further analysis showed that MdbHLH106L is able to bind the MdNHX1 promoter. Moreover, we also confirmed the interaction between MdbHLH106L and MdZAT10. In conclusion, MdbHLH106L participates in the salt-stress signaling pathway and may mediate salt tolerance by activating the MdNHX1 promoter and interacting with MdZAT10. Our findings may serve as a theoretical basis for further investigating molecular mechanisms of stress responses and for enriching genetic resources of stress-tolerant apple cultivars.

Melatonin, as a plant growth regulator, is involved in plant stress resistance. We studied the effects of different concentrations (0, 10, 50, 100, 150, and 200 µmol · L−1) of melatonin on the growth and physiological characteristics of strawberry under cadmium (Cd) stress. The results represented that the growth of strawberry seedlings was inhibited under Cd stress, and the seedling biomass, chlorophyll content and the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) decreased. These toxic effects were, however, effectively remediated by exogenous melatonin pretreatment. Melatonin treatment slowed the inhibitory effect of seedling growth and significantly increased seedling biomass, chlorophyll content, antioxidant enzyme activities, and soluble protein levels in leaves and roots compared with the control. Melatonin treatment also reduced the malondialdehyde (MDA) content and the oxidative stress reactions, increased the anthocyanin content, and slowed the senescence rate, thereby reducing the toxic effects of Cd on strawberries. This indicates that foliar application of melatonin can effectively remediate the adverse effects of Cd on strawberry seedlings; the greatest effect was obtained with melatonin concentration of 100 µmol · L−1.

Salt stress is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus × domestica). In this study, we demonstrate that a salt-responsive MYB transcription factor (TF), designated as MdMYB63, promotes survival under salt stress. Overexpression of MdMYB63 in apple calli significantly enhanced salt tolerance. Screening of the AP2/ERF family of TFs identified MdERF106 as an interaction partner of MdMYB63. Further analyses showed that the MdMYB63–MdERF106 complex significantly promotes the expression of downstream MdSOS1, thereby improving the Na+ expulsion and salt tolerance of apple. These functional analyses of MdMYB63 have provided valuable insights into the regulatory network of salt tolerance, and lay a theoretical foundation for the cultivation of new salt-tolerant apple varieties.

Phloridzin is a phenolic acid associated with the genus Malus (Mill.) that causes apple replant disease. The removal of phloridzin is of great significance to alleviate this condition, and hence bi...

A two-year field experiment was carried out in order to study the occurrence degree and mechanism of apple replant disease (ARD) in the apple orchards with different soil textures. So we can adopt appropriate control measures according to the severity of ARD. Healthy two-year-old seedlings with consistent growth were selected, of which the root stock was T337 and the scion was Yanfu 3. There were significant differences in biomass between methyl bromide fumigation and replanted treatments, and the difference was the largest in clay loam, followed by sandy loam, and loam, which verified ARD in clay loam was most serious, followed by sandy loam and loam. Based on high-throughput sequencing of fungi in soil samples, fungal richness and diversity were the highest in clay loam, followed by sandy loam, and loam. The relative abundance of Fusarium in SX, SL, FX, FL, WX and WL was 7.33%, 19.32%, 2.70%, 4.24%, 10.71% and 23.87%, respectively. Based on Real-time quantitative analysis, there were significant differences in the number of Fusarium oxysporum and Fusarium solani between methyl bromide fumigation and replanted treatments, i.e., clay loam > sandy loam > loam. Fusarium was the main pathogen causing ARD. This shows that ARD is the most serious under replanted clay loam condition. High-throughput sequencing technology was used to prove the difference in Fusarium was one of the important reasons for ARD under different soil textures. This technology provides a new idea for the prevention and control of ARD.

Summary Methylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured ‘Fuji’ apples and found that differences in the transcript levels of MdMYB1, which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (−1246 to −780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA‐directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4‐binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway.

Sugar content and cold tolerance are important apple (Malus × domestica) characteristics targeted by breeding programs. Here, a vacuolar membrane sucrose transporter, MdSWEET16, was cloned and characterized. A phylogenetic tree analysis found that MdSWEET16 was on the same polygenetic branch as Arabidopsis thaliana SWEET16 and SWEET17. MdSWEET16 was located on chromosome 2 and consists of six exons and five introns. The recombinant protein was obtained by prokaryotic induction, and the amino acid sequence and transmembrane domain were analyzed by bioinformatics. The encoded ~ 30-kDa protein has seven transmembrane domains and is localized on the tonoplasts of ‘Orin’ callus protoplasts. The expression of MdSWEET16 changed in response to sucrose and low temperature in ‘Orin’ calli. In addition, we also analyzed the expression level of MdSWEET16 during different fruit developmental stages using qRT-PCR. MdSWEET16 was highly expressed in young fruit, and its expression level during fruit development was significantly negatively correlated with sucrose content as assessed by a quantitative fluorescence analysis. Overexpression of MdSWEET16 in ‘Orin’ calli could reduce their sucrose content but increased their cold tolerance compared with MdSWEET16 RNA interference calli, which indicated that MdSWEET16 is involved in the sucrose transport and cold tolerance of apple. MdSWEET16’s expression level was significantly correlated with the sucrose content and low temperature, and was induced by sucrose and low temperatures. Overexpression of MdSWEET16 increased ‘Orin’ calli cold tolerance. The prokaryotic expression of its recombinant protein can be used to further study the function of the MdSWEET16 protein in apple.

The WRKY transcription factors play key roles in plant growth and abiotic stress responses; however, the molecular mechanisms behind their involvement in anthocyanin biosynthesis are still unclear. In our study, we identified a Leu zipper motif and a WRKY domain in MdWRKY40 protein. Phylogenetic tree analysis showed that MdWRKY40, AtWRKY18 and AtWRKY40 were on the same evolutionary branch and were Group IIa WRKY proteins. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that MdWRKY40 could interact with itself to form homodimers. Overexpressing MdMYB111 in red-fleshed callus inhibited the expression of MdANS and decreased the anthocyanin content. EMSA assay showed that MdMYB111 and MdWRKY40 could bind the MRE and the W box, respectively, in the MdANS promoter. Overexpressing MdWRKY40 in red-fleshed callus did not affect the expression of MdANS or the anthocyanin content. However, overexpressing MdWRKY40 in callus overexpressing MdMYB111 weakened the inhibitory effect of MdMYB111 on anthocyanin biosynthesis. Knocking out the Leu zipper motif of MdWRKY40 (LLSMdWRKY40) prevented its self-interaction, and knocking out C-x5-C sequence of MdWRKY40 (LCSMdWRKY40) prevented it from binding to W box. It did not weaken the inhibitory effect of MdMYB111 on anthocyanin biosynthesis when overexpressing LCSMdWRKY40 or LLSMdWRKY40 in callus overexpressing MdMYB111. Thus, MdMYB111 and MdWRKY40 may play important roles in the anthocyanin biosynthetic pathway. MdWRKY40 interacts with itself to form homodimers by the Leu zipper motif at the N-terminal, and it binds two W boxes distantly separated in the MdANS promoter in the presence of C-x5-C sequence. MdMYB111 binds the MRE in the looped region induced by MdWRKY40. In addition, it weakens the inhibitory effect of MdMYB111 on expression of MdANS and anthocyanin biosynthesis when overexpressing MdWRKY40 in callus overexpressing MdMYB111.

Ozone (O3) is an adverse environmental factor posing damage to ornamental plants. Thus, it is important to seek an effective way of enhancing plant tolerance to O3-induced damage. Methyl jasmonate (MJ) and melatonin (MT) are plant growth regulators (PGRs) involved in plant abiotic stress responses. In this study, compared with the control group of plants without ozone, the influence of exogenous MJ (0, 10, 50, 100, and 150 μM) and MT (0, 0.1, 0.5, 2.5, and 12.5 μM) on the resistance of Malus crabapple ‘Hong Jiu’ was evaluated under O3 stress (100 ± 10 nL/L for 3 h). Our data revealed that levels of MDA were significantly enhanced following O3 treatment compared with plants without O3. O3 induced the activities of antioxidant enzymes and the accumulation of non-enzymatic antioxidants. While lower malondialdehyde (MDA) content, greater activities of antioxidant enzymes, and higher levels of soluble protein and non-enzymatic antioxidants were observed in PGRs-pretreated plants than in non-PGRs-pretreated plants under O3 stress. Based on the above results and air pollution tolerance index (APTI), an exogenous supply of MJ and MT to Malus crabapple ‘Hong Jiu’ seedlings was protective for O3-induced toxicity. The present study provides new insights into the mechanisms of MJ and MT amelioration of O3-induced oxidative stress damages in Malus crabapple ‘Hong Jiu.’

Light is an important environmental factor affecting plant growth and development. Additionally, HY5 is a central factor that coordinates light signal transduction and regulates the expression of flower color-related genes. However, there are few reports describing the co-regulation of apple fruit coloration by MdHY5 and MYB transcription factors. In this study, we detected a light-inducible gene, MdMYBDL1, which encodes a MYB-like domain and is homologous to AtMYBD in Arabidopsis thaliana. Moreover, we observed that MdHY5 binds to the G-box element of the MdMYBDL1 promoter to upregulate expression. The overexpression of MdMYBDL1 enhanced anthocyanin accumulation in apple calli and inhibited the expression of MdMYB16 and its homolog, MdMYB308. Furthermore, MdMYB16 can form a dimer with MdMYB308 and functions as a negative regulator of anthocyanin biosynthesis. Interestingly, MdMYB16 and MdMYB308 promoter activities were inhibited by MdMYBDL1 and MdHY5. These findings imply that MdHY5 responds to light signals and functions upstream of different types of MYB transcription factors, ultimately regulating anthocyanin accumulation in apples.

Malus sieversii and its red-fleshed variant (M. sieversii f. niedzwetzkyana) can provide important resources for producing cider. This not only helps to protect wild germplasm resources, but also extends the capabilities of the apple industry. So the present study aimed to determine the changes in nutritional quality during the brewing process to obtain the optimal fermentation time. The samples were taken periodically and we use HPLC, HPCE, GC–MS, ICP-MS and other methods to determine the nutritional components of red-fleshed cider. The results showed that the contents of 24 polyphenols, mineral elements and organic acids were in dynamic change during fermentation and types and contents of 16 kinds of amino acids were decreased. At a fermentation temperature of 25–27 °C and using a standard yeast inoculation, the optimal fermentation time of red-fleshed cider being 8 days, for the transformation of sugars to alcohols can be done thoroughly and retain more nutrients. The fermentation process for red-fleshed cider consisted mainly of a number of different complex microbial and biochemical reactions.

Background Fruit peel colour is an important agronomic trait for fruit quality. Cytosine methylation plays an important role in gene regulation. Although the DNA methylation level of a single gene is important to affect the phenotype of mutation, there are large unknown of difference of the DNA methylation in plant and its mutants. Results Using bisulfite sequencing (BS-Seq) and RNA-sequencing (RNA-Seq), we analysed three deep-red-skinned apple (Malus × domestica) mutants (Yanfu 3, YF3; Yanfu 8, YF8; Shannonghong, SNH) and their lighter-skinned parents (Nagafu 2, NF2; Yanfu 3, YF3; Ralls, RL) to explore the different changes in methylation patterns associated with anthocyanin concentrations. We identified 13,405, 13,384, and 10,925 differentially methylated regions (DMRs) and 1987, 956, and 1180 differentially expressed genes (DEGs) in the NF2/YF3, YF3/YF8, and RL/SNH comparisons, respectively. And we found two DMR-associated DEGs involved in the anthocyanin pathway: ANS (MD06G1071600) and F3H (MD05G1074200). These genes exhibited upregulated expression in apple mutants, and differences were observed in the methylation patterns of their promoters. These results suggested that both the regulatory and structural genes may be modified by DNA methylation in the anthocyanin pathway. However, the methylation of structural genes was not the primary reason for expression-level changes. The expression of structural genes may be synergistically regulated by transcription factors and methylation changes. Additionally, the expression of the transcription factor gene MYB114 (MD17G1261100) was upregulated in the deep-red-skinned apple. Conclusion Through the analysis of global methylation and transcription, we did not find the correlation between gene expression and the DNA methylation. However, we observed that the upregulated expression of ANS (MD06G1071600) and F3H (MD05G1074200) in apple mutants results in increased anthocyanin contents. Moreover, MYB114 (MD17G1261100) is likely another regulatory gene involved in apple coloration. Our data provided a new understanding about the differences in formation of apple colour mutants. Electronic supplementary material The online version of this article (10.1186/s12864-019-5499-2) contains supplementary material, which is available to authorized users.

In many plants, anthocyanin biosynthesis is affected by environmental conditions. Ultraviolet-B (UV-B) radiation promotes anthocyanin accumulation and fruit coloration in apple skin, whereas high temperature suppresses these processes. In this study, we characterized a B-box transcription factor, MdCOL4, from 'Fuji' apple, and identified its role in anthocyanin biosynthesis by overexpressing its encoding gene in apple red callus. The expression of MdCOL4 was reduced by UV-B, but promoted by high temperature. We explored the regulatory relationship between heat shock transcription factors (HSFs) and MdCOL4, and found that MdHSF3b and MdHSF4a directly bound to the heat shock element cis-element of the MdCOL4 promoter. MdCOL4 interacted with MdHY5 to synergistically inhibit the expression of MdMYB1, and MdCOL4 directly bound to the promoters of MdANS and MdUFGT, which encode genes in the anthocyanin biosynthetic pathway, to suppress their expression. Our findings shed light on the molecular mechanism by which MdCOL4 suppresses anthocyanin accumulation in apple skin under UV-B and high temperature.

The transition from vegetative to reproductive growth is the most dramatic phase change in plants. To better understand the molecular regulation of floral transition and flower development in red-fleshed apple (Malus sieversii f. niedzwetzkyana), we isolated and characterized a floral MADS-box gene, MdAGL24-like, which shares sequence similarity with AGAMOUS-LIKE 24 (AGL24) from other species. Spatial expression analysis showed that MdAGL24-like dynamically expressed in flowers, followed by roots and fruits. Subcellular localization analysis indicated that, like other transcript factors, MdAGL24-like was localized in the nucleus. Protein interaction analysis showed that MdAGL24-like could interact with MdSOC1 and MdAP1 in vivo and in vitro. MdAGL24-like and MdSOC1 could increase each other’s expression by binding the CArG motifs in their promoters. Unlike MdSOC1, MdAGL24-like might indirectly promote the expression of MdLFY by upregulating the expression of MdSOC1. Ectopic expression of MdAGL24-like in wild-type Arabidopsis induced early flowering like the phenotypes induced by other AGL24 genes. Similar to AGL24 in Arabidopsis, MdAGL24-like could rescue the late-flowering phenotype of the agl24 mutant to some extent. These results help clarify the molecular mechanism underlying flowering and provide a means of shortening the juvenile period in red-fleshed apples and other fruit trees.

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.

Flavonoid content, which is an important indicator of the nutritional value of fruits and vegetables, directly determines the marketability of many fruit crops, including apple (Malus domestica). Brassinosteroids (BRs) are steroid hormones that affect flavonoid biosynthesis in plants, but the underlying regulatory mechanism remains unclear. In this study, treatments with brassinolide (the most active BR) and brassinazole (a BR biosynthesis inhibitor) decreased and increased, respectively, the flavonoid, anthocyanin, and proanthocyanidin (PA) content in red-fleshed apple seedlings and calli. We subsequently demonstrated that a BZR (BRI1-EMS-suppressor (BES)/brassinazole-resistant) family transcription factor, MdBEH2.2, participates in BR-regulated flavonoid biosynthesis. Specifically, MdBEH2.2 inhibits the accumulation of flavonoids, anthocyanins, and PAs in apple seedlings; however, brassinazole treatment weakens the inhibitory effect. Additionally, we confirmed that a BR-induced MYB TF, MdMYB60, interacts with MdBEH2.2. The resulting MdBEH2.2–MdMYB60 complex further enhances the inhibitory effect of MdBEH2.2 or MdMYB60 on the transcription of flavonoid biosynthesis-related genes. These results indicate that brassinolide decreases flavonoid content through the MdBEH2.2–MdMYB60 regulatory module. Our findings further clarify the molecular mechanism mediating the regulation of flavonoid biosynthesis by BR signals in horticultural crops.

Apple (Malus domestica Borkh.) is one of the most widely produced and economically important fruits in temperate regions. Fruit color development in apple is a major focus for both breeders and researchers as consumers associate brightly colored red apples with ripeness and a good flavor. In recent years, great progress has been made in the research of apple fruit color development, but its development mechanism has not been systematic dissected from the aspects of genetics, transcription or environmental factors. Here, we summarize research on the coloration of apple fruit, including the development of important genomic databases to identify important genomic regions and genes, genetic and transcriptional factors that regulate pigment accumulation, environmental factors that affect anthocyanin synthesis, and the current breeding progress of red-skinned and red-fleshed apples. We describe key transcription factors, such as MYB, bHLH, and WD40, which are involved in the regulation of anthocyanin synthesis and fruit color development in apple. We also discuss the regulation of apple color by external environmental factors such as light, temperature, and water. The aim of this review is to provide insights into the molecular mechanisms underlying anthocyanin biosynthesis in apple. This information will provide significant guidance for the breeding of high-quality red-skinned and red-fleshed apple varieties.

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.

Background Apple replant disease (ARD) has been reported from all major fruit-growing regions of the world, and is often caused by biotic factors (pathogen fungi) and abiotic factors (phenolic compounds). In order to clarify the proteomic differences of Fusarium moniliforme under the action of phloridzin, and to explore the potential mechanism of F. moniliforme as the pathogen of ARD, the role of Fusarium spp. in ARD was further clarified. Methods In this paper, the quantitative proteomics method iTRAQ analysis technology was used to analyze the proteomic differences of F. moniliforme before and after phloridzin treatment. The differentially expressed protein was validated by qRT-PCR analysis. Results A total of 4535 proteins were detected, and 293 proteins were found with more than 1.2 times (PP Conclusions This study is the first to perform quantitative proteomic investigation by iTRAQ labeling and LC-MS/MS to identify differentially expressed proteins in F. moniliforme under phloridzin conditions. The results confirmed that F. moniliforme presented a unique protein profile that indicated the adaptive mechanisms of this species to phloridzin environments. The results deepened our understanding of the proteome in F. moniliforme in response to phloridzin inducers and provide a basis for further exploration for improving the efficiency of the fungi as biocontrol agents to control ARD.

Ozone (O3), an oxidizing toxic air pollutant, is ubiquitous in industrialized and developing countries. To understand the effects of O3 exposure on apple (Malus) and to explore its defense mechanisms, we exposed 'Hongjiu' crabapple to O3 and monitored its responses using physiological, transcriptomics, and metabolomics analyses. Exposure to 300 nL L-1 O3 for 3 h caused obvious damage to the leaves of Malus crabapple, affected chlorophyll and anthocyanin contents, and activated antioxidant enzymes. The gene encoding phospholipase A was highly responsive to O3 in Malus crabapple. McWRKY75 is a key transcription factor in the response to O3 stress, and its transcript levels were positively correlated with those of flavonoid-related structural genes (McC4H, McDFR, and McANR). The ethylene response factors McERF019 and McERF109-like were also up-regulated by O3. Exogenous methyl jasmonate (MeJA) decreased the damaging effects of O3 on crabapple and was most effective at 200 μmol L -1. Treatments with MeJA altered the metabolic pathways of crabapple under O3 stress. In particular, MeJA activated the flavonoid metabolic pathway in Malus, which improved its resistance to O3 stress.

Flavonoids play essential roles in human health. Apple (Malus domestica Borkh.), one of the most widely produced and economically important fruit crops in temperate regions, is a significant source of flavonoids in the human diet and is among the top nutritionally rated and most widely consumed fruits worldwide. Epidemiological studies have shown that the consumption of apples, which are rich in a variety of free and easily absorbable flavonoids, is associated with a decreased risk of various diseases. However, apple production is challenged by serious inbreeding problems. The narrowing of the hereditary base has resulted in apples with poor nutritional quality and low flavonoid contents. Recently, there have been advances in our understanding of the roles that Malus sieversii (Ledeb.) M.Roem has played in the process of apple domestication and breeding. In this study, we review the origin of cultivated apples and red-fleshed apples, and discuss the genetic diversity and construction of the core collections of M. sieversii. We also discuss current research progress and breeding programs on red-skinned and red-fleshed apples and summarize the exploitation and utilization of M. sieversii in the breeding of high-flavonoid, and red-fleshed apples. This study highlights a valuable pattern of horticultural crop breeding using wild germplasm resources. The future challenges and directions of research on the molecular mechanisms of flavonoid accumulation and high-flavonoid apple breeding are discussed., Molecular biology: Making apples even healthier They say that an apple a day keeps the doctor away, but the breeding of new varieties of red-fleshed apples with higher flavonoid contents could make this widely-consumed fruit even healthier. Apples are one of the richest dietary sources of flavonoids—metabolites credited with reducing the risk of various diseases—but during the process of domestication, the genetic diversity and nutritional quality of apples have decreased. In this review, Xue-Sen Chen at Shandong Agricultural University in China and colleagues track the evolution of modern cultivated apples from their ancestor, Malus sieversii, and outline recent developments in our understanding of the molecular pathways underpinning flavonoid synthesis. They also consider how M. sieversii could be used to create new varieties of tasty red-skinned and red-fleshed apples, enriched with beneficial metabolites.

Improving the cold resistance of plants is important, because their growth and development are negatively affected by cold stress. In this study, exogenous applications of methyl jasmonate could enhance the cold resistance of ‘Orin’ apple (Malus × domestica) calli by increasing the expression levels of the cold-signal response genes MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3. In addition, yeast two-hybrid and pull-down assays demonstrated that MdCIbHLH1 interacts with MdJAZ1/4 and MdMYC2 in vitro and in vivo. Protein degradation experiments demonstrated that the stability of the MdJAZ1/4 proteins were affected by the application of exogenous methyl jasmonate, which was followed by their degradation by the 26S proteasome. MdJAZ1/4 act as repressors, binding MdMYC2 in the jasmonate-signaling pathway. The overexpression of MdMYC2 in ‘Orin’ calli increased the expression levels of MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3, resulting in an increased freeze tolerance. Furthermore, the overexpression of MdJAZ1 or MdJAZ4 in transgenic red-fleshed apple calli weakened the promotive effect of MdMYC2 on cold tolerance. Yeast one-hybrid and chromatin immunoprecipitation-PCR analyses showed that MdMYC2 could bind the G-box element found in the MdCBF1 promoter. Thus, jasmonate may function as a critical upstream signal in the ICE–CBF/DREB1 pathway to positively regulate apple freeze tolerance.

In plants, flavonoids play critical roles in resistance to biotic and abiotic stresses, and contribute substantially to the quality, flavor, and nutritional quality of many fruit crops. In apple (Malus × domestica), inbreeding has resulted in severe decreases in the genetic diversity and flavonoid content. Over the last decade, we have focused on the genetic improvement of apple using wild red-fleshed apple resources (Malus sieversii f. niedzwetzkyana). Here, we found that the MYB transcription factors (TFs) involved in the synthesis of proanthocyanidins can be classified into TT2 and PA1 types. We characterized a PA1-type MYB transcription factor, MdMYBPA1, from red-fleshed apple and identified its role in flavonoid biosynthesis using overexpression and knockdown-expression transgenes in apple calli. We explored the relationship between TT2- and PA1-type MYB TFs, and found that MdMYB9/11/12 bind the MdMYBPA1 promoter. In addition, MdMYBPA1 responded to low temperature by redirecting the flavonoid biosynthetic pathway from proanthocyanidin to anthocyanin production. In binding analyses, MdbHLH33 directly bound to the low-temperature-responsive (LTR) cis-element of the MdMYBPA1 promoter and promotes its activity. In addition, the calli expressing both MdMYBPA1 and MdbHLH33, which together form a complex, produced more anthocyanin under low temperature. Our findings shed light on the essential roles of PA1-type TFs in the metabolic network of proanthocyanidin and anthocyanin synthesis in plants. Studies on red-fleshed wild apple are beneficial for breeding new apple varieties with high flavonoid contents.

In plants, flavonoids are important secondary metabolites that contribute to the nutritional quality of many foods. Apple is a popular and frequently consumed food because of its high flavonoid content. In this study, flavonoid composition and content were detected and compared between red- and white-fleshed apples in a BC1 hybrid population using ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry. Transcriptomic analysis of the red- and white-fleshed apples was then performed using RNA-seq technology. By screening differentially expressed genes encoding transcription factors, we unearthed a WRKY-family transcription factor designated MdWRKY11. Overexpression of MdWRKY11 promoted the expression of F3H, FLS, DFR, ANS, and UFGT and increased the accumulation of flavonoids and anthocyanin in apple calli. Our findings explored the novel role of MdWRKY11 in flavonoid biosynthesis and suggest several other genes that may also be potentially involved. This provides valuable inform...

As cold stress greatly affects plant growth and development, understanding the mechanisms underlying cold tolerance in plants is important. In this study, we analyzed the expression levels of apple (Malus domestica) MdbHLH33 and MdCBF1–5 by semi-quantitative PCR after exposure to 4 °C for different amounts of time and generated evolutionary trees for MdbHLH33 and the MdCBFs. Overexpressing MdbHLH33 pro-GUS in ‘Orin’ callus, indicated that transgenic callus had higher GUS activity and was more deeply stained at 4 °C than at 25 °C. Subcellular localization showed that MdbHLH33 was located in the nucleus. Overexpressing MdbHLH33 in ‘Orin’ callus increased the expression level of MdCBF2, MdCOR15A-1, and MdCOR15A-2, and resulted in increased cold tolerance. EMSA and Chip-PCR analysis showed that MdbHLH33 could bind the LTR cis-acting element found in the MdCBF2 promoter. Overexpressing MdCBF2 in ‘Orin’ callus indicated that MdCBF2 could also increase the expression level of MdCOR15A-1 and MdCOR15A-2 and improve cold tolerance; we also found that transgenic callus overexpressing MdCBF2 had reduced MdCBF1 and MdCBF5 expression and increased MdCBF3 and MdCBF4 expression. Overall, these results show that MdbHLH33 can regulate the expression of MdCBF2 and improve the cold tolerance of transgenic callus.

In this study, the effects of phloridzin, phloretin and benzoic acid on the root proteome of Malus hupehensis Rehd seedlings under the sandy culture conditions were investigated. The applied concentrations of phloridzin, phloretin and benzoic acid were the measured concentrations in soil. Compared with the control group, a total of 16 differentially expressed protein spots (10 up-regulated and 6 down-regulated), 13 differentially expressed protein spots (4 up-regulated and 9 down-regulated) and 9 differentially expressed protein spots (3 up-regulated and 6 down-regulated) were detected in the root systems after the M. hupehensis Rehd seedlings were treated with phloridzin, phloretin or benzoic acid, respectively. The identified protein spots could be classified into 8 functional groups as follows: signal transduction-related proteins, pathogenesis-related proteins, metabolism-related proteins, free radical scavenging proteins, stress resistance-related proteins, cytoskeleton-related proteins, allergen-related proteins and proteins with unknown functions. Under the stresses of phloridzin, phloretin and benzoic acid treatments, the expression levels of defense system-related proteins and free radical scavenging proteins in their root system were up-regulated, indicating that the phenolic compounds, such as phloridzin, phloretin and benzoic acid, could cause damaging effects on M. hupehensis Rehd seedlings, leading to the up-regulation of defense system-related proteins. This study further revealed that the action mechanisms by which phenolic compounds play their roles in apple replant disease (ARD), which could provide useful information for comprehensive control and prevention of ARD.

As a family of transcription factors, DNA binding with one figure (Dof) proteins play important roles in various biological processes in plants. Here, a total of 60 putative apple (Malus domestica) Dof genes (MdDof) were identified and mapped to different chromosomes. Chromosomal distribution and synteny analysis indicated that the expansion of the MdDof genes came primarily from segmental and duplication events, and from whole genome duplication, which lead to more Dof members in apples than in other plants. All 60 MdDof genes were classified into thirteen groups, according to multiple sequence alignment and the phylogenetic tree constructed of Dof genes from apple, peach (Prunus persica), Arabidopsis and rice. Within each group, the members shared a similar exon/intron and motif compositions, although the sizes of the MdDof genes and encoding proteins were quite different. Several Dof genes from the apple and peach were identified to be homologues based on their close synteny relationship, which suggested that these genes bear similar functions. Half of the MdDof genes were randomly selected to determine their responses to different stresses. The majority of MdDof genes were quite sensitive to PEG, NaCl, cold and exogenous ABA treatment. Our results suggested that MdDof family members may play important roles in plant tolerance to abiotic stress.

Nitrogen limitation stress is known to markedly promote anthocyanin accumulation and upregulate anthocyanin biosynthesis-related genes; however, the underlying molecular mechanisms have not been fully characterized. In this study, a callus was induced from the leaves of an R6 homozygous red-fleshed apple, the hybrid offspring of Malus sieversii f. niedzwetzkyana and Malus × domestica ‘Fuji.’ We analyzed the growth and accumulation of anthocyanin in callus under different nitrogen concentrations (0.01, 0.02, 0.04, and 0.06 M NO3 −). The results showed that the callus growth in the 0.01 M NO3 − condition was minimal, but its anthocyanin content was significantly elevated compared with those under the other nitrogen treatments. As the concentration of nitrogen increased from 0.01 to 0.04 M, the expression of the anthocyanin structural genes, MdCHS and MdDFR, and the transcription factor genes MdMYB9, MdMYB10, MdbHLH3, and MdbHLH33 were downregulated. On the contrary, the transcript levels of MdLOB52 and MdARF19 were increased by the nitrogen treatments. The overexpression of MdLOB52 inhibited anthocyanin accumulation in the callus. Subsequent yeast one-hybrid assays showed that MdARF19 can directly bind the promoter of MdLOB52, which may strengthen the activation of MdLOB52 and affect anthocyanin accumulation. Our findings provide new insights into the mechanisms by which the LOB genes respond to nitrogen to regulate anthocyanin biosynthesis in a red-fleshed apple callus.

This study examined the effects of postharvest storage conditions on the fruit quality of a new red-fleshed apple cultivar ('Meihong'). Mature 'Meihong' and 'Golden delicious' apples were exposed to room temperature, low temperature, and low temperature and 1-MCP, after which several fruit characteristics were evaluated (i.e., firmness, ethylene release rate, relative content of aroma components, phenolic compounds and antioxidant capacity, fruit softening-related enzyme activities, and related gene expression). Both 'Meihong' and 'Golden delicious' were ACS1-1/-2 heterozygotes, but the ethylene release rate in 'Meihong' fruits was lower than that in 'Golden delicious' fruits during storage. Therefore, 'Meihong' fruits are more conducive to storage. The low temperature storage with and without 1-MCP delayed fruit softening, decreased the ethylene release rate and ester aroma component content, and maintained total flavonoid and anthocyanin contents. Therefore, storage at low temperatures with 1-MCP or other preservatives may be useful for maintaining the 'Meihong' fruit quality.

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.

In this study, we explored the effects of short-term rotation and Trichoderma application on the plant biomass, root respiration rate, root protective enzymes of Malus hupehensis Rehd., and soil environment under replant conditions, to provide a basis for the prevention of the apple replant disease (ARD). In 2014, old apple orchard soils were treated by rotating peanut, Allium fistulosum L., fallow, and natural grass, and replanted apple orchard soil was used as the control. The soils of the different treatments were mixed well with Trichoderma at a concentration of 0.5% (w/w), and 6 kg of each treated soil was added to a pot (outer diameter, 27 cm; inner diameter, 23 cm; height, 18 cm). Uniform seedlings of Malus hupehensis Rehd. were transplanted to the pots on April 23, 2015. Two seedlings were planted in each pot, and 30 pots were prepared for each treatment. The samples were collected at the end of August, when there were visible differences in seedling growth among the treatments. The results showed that rotating Allium fistulosum L. mixed with Trichoderma could significantly improve the populations of bacteria and actinomycetes in the soil, which increased by 189.4% and 107.1%, respectively. The biomass of M. hupehensis significantly improved, and plant fresh weight, dry weight, height, and diameter increased by 186.3%, 205.9%, 58.8%, and 33.2%, respectively. In addition, the total fine root length, total fine root surface area, total fine root volume, and fine root tip number increased by 147.2%, 225.9%, 298.5%, and 331.3%, respectively. Compared with the control seedlings, the treated seedlings showed higher respiration rate of the root system, Superoxide Dismutase activity and Peroxidase activity by 131.0%, 46.3%, and 110%, respectively. Analysis of the terminal restriction fragment length polymorphism profiles showed that the treatment of short-term crop rotation and Trichoderma changed the soil fungal community structure; improved the Simpson index of the soil fungal community; and reduced the Shannon index, Pielou index, and Margalef index. The results suggest that short-term rotation of Allium fistulosum L. mixed with Trichoderma could alleviate ARD.

Exposure to high salt concentrations is one of the main stresses adversely affecting crop growth, development, and yield. In this study, a putative salt stress-responsive gene (MdMYB4) encoding an R2R3-MYB transcription factor was cloned and characterized. This gene belongs to the SG2 subgroup, and its C-terminal contains a conserved SG2 motif. The relative MdMYB4 expression levels in Malus × domestica Borkh. ‘Orin’ calli cultured under various NaCl concentrations were analyzed. Saline conditions upregulated the expression of MdMYB4, and the effects of the encoded transcription factor on salt tolerance were determined in MdMYB4-overexpressing (OE-MYB4) ‘Orin’ calli. The OE-MYB4 calli grew significantly better than the wild-type calli under salt stress conditions. Additionally, an analysis of the cis-acting elements in the MdNHX1 and MdSOS1 promoters revealed MYB-binding sites. However, the electrophoretic mobility shift assay results indicated that MdMYB4 can bind only to the MdNHX1 promoter. Moreover, the MdNHX1 expression levels were higher in OE-MYB4 calli than in wild-type calli at 150, 200, and 250 mM NaCl. In contrast, there were no significant changes to the MdSOS1 expression levels in OE-MYB4 calli. In conclusion, MdMYB4 is an R2R3-MYB transcription factor that mediates salt tolerance by activating the MdNHX1 promoter. Our findings may provide a theoretical basis for enriching the genetic resources associated with stress tolerance and for improving stress resistance in apple cultivars.

We investigated the effects of seaweed fertilizer on the physiological features of apple seedlings under continues cropping conditions. The six-leaf Malus hupehensis Rehd. seedlings were planted in replant soil with seaweed fertilizer applied at three rates: 5, 20, and 40 g kg− 1, and one control group with no seaweed fertilizer application. The results showed that the addition of seaweed fertilizer obviously increased the seedling plants growth. The rate of photosynthesis, chlorophyll (a + b) content, and activities of protective enzymes (superoxide dismutase, ascorbate peroxidase, and catalase), were 78%, 20%, 23%, 45% and 144% higher, respectively, in the seedlings treated with 40 g kg− 1 of seaweed fertilizer than in the control group. Furthermore, plants in the 40 g kg− 1 seaweed fertilizer treatment group had H2O2, O2−, malondialdehyde and proline contents that were 32%, 60%, 56% and 38% lower, respectively, than in the control group. Finally, the number of bacteria, fungi, and the ratio of bacteria:fungi in the replant soil with 40 g kg− 1 of seaweed fertilizer applied were 172%, 67% and 150% higher, respectively, than in the soil without seaweed fertilizer application. Overall, seaweed fertilizer application promoted plant growth, increased the rate of photosynthesis, activated antioxidant enzymes, decreased lipid peroxidation, improved the soil nutrient, and regulated the soil microorganisms. As a result of these changes, the treated seedlings did not exhibit the symptoms of apple replant disease.

We researched the effects of methyl jasmonate, abscisic acid, and a combination of the two on anthocyanin metabolism in callus material induced from the leaves of a red-fleshed apple individual, which was a hybrid offspring of Malus sieversii f. niedzwetzkyana and Malus domestica cv. ‘Fuji’. The results showed that methyl jasmonate promoted anthocyanin accumulation with increasing methyl jasmonate concentration in the range of 10−6 to 10−3 mol/L. Abscisic acid significantly inhibited anthocyanin biosynthesis, and its inhibitory effect was highest at 6 μmol/L among all treatments. In addition, methyl jasmonate alleviated the inhibitory effect of abscisic acid on anthocyanin biosynthesis. Gene expression analysis indicated that methyl jasmonate induced the expression of MdMYB9 and MdMYB10, which are positive regulator of anthocyanin biosynthesis, and inhibited MdMYB11 expression. It also upregulated the expression of the anthocyanin structural genes MdCHS, MdF3H and MdUFGT. Abscisic acid inhibited the expression of MdMYB3 and MdMYB10, as well as the expression of the structural genes MdF3H, MdDFR, MdLDOX and MdUFGT. Methyl jasmonate relieved the inhibition by abscisic acid by decreasing the expression of MdMYB11 and MdMYB16, and increasing the expression of the anthocyanin regulatory genes MdMYB3, MdMYB9 and MdMYB10, and the structural genes MdCHS, MdDFR, MdF3H and MdUFGT. Thus, we elucidated the roles of methyl jasmonate and abscisic acid in the regulation of anthocyanin production at the gene transcription level.

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1-5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

Volatile compounds have a tremendous impact on fruit quality. We evaluated the volatile compound profiles of ripening wild apple fruit (10 Malus baccata accessions and three Malus prunifolia accessions) in the National Field Genebank for Hardy Fruits at Gongzhuling, China. Alcohols, esters, aldehydes, terpenes, hydrocarbons, ethers, heterocycles, carboxylic acids, and ketones were detected in the M. baccata and M. prunifolia fruit, with the first four being the main volatile compounds present. Of the 92 volatiles detected, esters were the most diverse (49 compounds). This wide range of abundant volatile compounds suggests that M. prunifolia is a good resource for breeding apple cultivars with novel and interesting flavors. The M. baccata accession ‘Zhaai Shandingzi’ and the M. prunifolia accession ‘Bai Haitang’ had the widest range of volatile compounds and the highest volatile compound contents of the accessions examined, and will therefore be good breeding materials for developing commercial lines with enhanced flavor and for widening the genetic diversity. The number of different ester compounds present was significantly positively correlated (r = 0.877) with the cube root of the weight of an individual ripe fruit. Principal component analysis (PCA) showed that the contents of ester compounds could be used to distinguish between M. baccata and M. prunifolia species. Therefore, ester compounds could be used as a reference of parental choice in apple breeding.

Ethylene and xyloglucan endotransglucosylase/hydrolase (XTH) genes were important for fruit ripening and softening in 'Taishanzaoxia' apple. In this study, we found it was ACS1-1/-1 homozygotes in 'Taishanzaoxia' apple, which determined the higher transcription activity of ACS1. XTH1, XTH3, XTH4, XTH5, and XTH9 were mainly involved in the early fruit softening independent of ethylene, while XTH2, XTH6, XTH7, XTH8, XTH10, and XTH11 were predominantly involved in the late fruit softening dependent on ethylene. Overexpression of XTH2 and XTH10 in tomato resulted in the elevated expression of genes involved in ethylene biosynthesis (ACS2, ACO1), signal transduction (ERF2), and fruit softening (XTHs, PG2A, Cel2, and TBG4). In summary, the burst of ethylene in 'Taishanzaoxia' apple was predominantly determined by ACS1-1/-1 genotype, and the differential expression of XTH genes dependent on and independent of ethylene played critical roles in the fruit ripening and softening. XTH2 and XTH10 may act as a signal switch in the feedback regulation of ethylene signaling and fruit softening.

Apple replant disease (ARD) has been reported from all major fruit-growing regions of the world, and is often caused by biotic (pathogen fungi) and abiotic (phenolic compounds) factors. However, how phenolic compounds affect pathogen fungi is not yet fully understood. The aims of this study were to investigate whether the Fusarium moniliforme ( F. moniliforme , a soil-borne pathogen of apple) could be labeled with fluorescent silicon carbide (SiC) quantum dots for a long-term tracing, and to observe the effect of phloridzin on the growth and conidia morphology of F. moniliforme with the help of fluorescence labeling with SiC quantum dots. The phloridzin was set to three concentrations (0 mM, 0.5 mM and 1.0 mM). Results showed that F. moniliforme could be labeled with SiC quantum dots and that the fluorescence in the mycelium could be observed for at least 40 days. The fluorescent images during the labeling process showed that F. moniliforme growth and division were slower when there was no phloridzin. The growth and division of F. moniliforme were fast with the help of phloridzin, and the growth and division were faster in the 1.0 mM phloridzin than in the 0.5 mM phloridzin. These results suggested that SiC quantum dots could successfully label F. moniliforme for a long-term tracing, and phloridzin could promote the growth and conidia division of F. moniliforme under the experimental concentration.

Methyl jasmonate (MeJA) has significant effects on the apple ripening process, but little is known about the molecular mechanisms by which MeJA acts in the regulation of this complicated process. To address this question, transcriptome profiles of MeJA-treated apples and mock-treated controls were generated using RNA-sequencing technology and then compared. A total of 1092 transcripts changed significantly in response to MeJA, with 684 up-regulated and 408 down-regulated. In MeJA-treated apples, genes involved in anthocyanin biosynthesis and transport were commonly up-regulated. The up-regulated genes also included genes in the biosynthetic and signal transduction pathways for the hormones jasmonic acid and ethylene. In contrast, exogenous MeJA generally down-regulated genes associated with auxin signal transduction. Furthermore, the transcript levels of 10 jasmonic acid biosynthetic genes, 7 selected ethylene biosynthetic and signal transduction genes, 5 selected auxin signal transduction genes, and 8 selected anthocyanin biosynthetic and transport genes were confirmed via real-time qPCR. These results suggest that MeJA promotes apple ripening, likely by activating ethylene signaling and inhibiting auxin action. Because MeJA-treated apples have previously been documented to have high anthocyanin concentrations, the molecular mechanism underlying this phenomenon is also discussed.

Anthocyanins are biosynthesized on the cytosolic surface of the endoplasmic reticulum and then transported into the vacuole for storage. Glutathione S-transferases (GSTs) are considered to be responsible for the transport of anthocyanins into the vacuole. However, the regulatory mechanisms of GSTs in plants are still unclear. Here, we performed a genome-wide analysis and identified 69 GST genes in apple. The expression of MdGSTF6 was positively correlated with the anthocyanin content (r = 0.949) during ‘Yanfu 8’ fruit development. The overexpression of MdGSTF6 in the Arabidopsis thaliana tt19 mutant resulted in seedlings of 35S::MdGSTF6-GFP/tt19 that could accumulate anthocyanin and rescue its phenotype, suggesting that MdGSTF6 was an anthocyanin transporter. The silencing of MdGSTF6 affected anthocyanin accumulation in apple fruit. Moreover, the knockdown of MdGSTF6 by RNA interference in cultured ‘Gala’ seedlings inhibited anthocyanin accumulation. The interaction experiments showed that MdMYB1 could bind directly to the MdGSTF6 promoter to transcriptionally activate its expression. Collectively, our results demonstrate that MdGSTF6 encodes an important GST transporter of anthocyanins in apple fruit and provide evidence for the associated regulatory mechanisms. Therefore, MdMYB1 can not only regulate anthocyanin synthesis, but also control the transport of anthocyanin in apples. This information may be useful for further clarifying the regulation of anthocyanin transport in apple., Intracellular transport: Identifying how apple pigment is sent for storage The mystery of how pigmenting anthocyanins are transported to intracellular storage is starting to be revealed. Alongside giving fruit their color, anthocyanins confer benefits to plant and human health. Glutathione S-transferases (GSTs) are responsible for transporting anthocyanins to storage vacuoles, yet research is lacking on how GSTs are regulated. Xuesen Chen, from China’s Shandong Agricultural University, and his team analyzed the activity of 23 GST genes in apple, finding one, MdGSTF6, was most highly active in the fruit-coloration stage of apple development. MdGSTF6 expression correlated with fruit anthocyanin levels and also restored anthocyanin levels in transport-suppressed plants, confirming its function. Chen’s team then discovered that MdGSTF6 expression is activated by the protein MdMYB1. The MdMYB1 gene is the major regulatory gene in anthocyanin synthesis, and is now revealed to also influence their transport.

Auxin signaling, which is crucial for normal plant growth and development, mainly depends on ARF–Aux/IAA interactions. However, little is known regarding the regulatory effects of auxin signaling on anthocyanin metabolism in apple (Malus domestica). We investigated the functions of MdARF13, which contains a repression domain and is localized to the nucleus. This protein was observed to interact with the Aux/IAA repressor, MdIAA121, through its C-terminal dimerization domain. Protein degradation experiments proved that MdIAA121 is an unstable protein that is degraded by the 26S proteasome. Additionally, MdIAA121 stability is affected by the application of exogenous auxin. Furthermore, the overexpression of MdIAA121 and MdARF13 in transgenic red-fleshed apple calli weakened the inhibitory effect of MdARF13 on anthocyanin biosynthesis. These results indicate that the degradation of MdIAA121 induced by auxin treatment can release MdARF13, which acts as a negative regulator of the anthocyanin metabolic pathway. Additionally, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays confirmed that MdMYB10 interacts with MdARF13. A subsequent electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that MdARF13 directly binds to the promoter of MdDFR, which is an anthocyanin pathway structural gene. Interestingly, chromatin immunoprecipitation–quantitative real-time PCR results indicated that the overexpression of MdIAA121 clearly inhibits the recruitment of MdARF13 to the MdDFR promoter. Our findings further characterized the mechanism underlying the regulation of anthocyanin biosynthesis via Aux/IAA–ARF signaling., A green light for red apples Insights into a hormonal signaling pathway controlling apple coloration could help growers to consistently produce eye-catching fruit. Auxins are signaling molecules that regulate many important metabolic pathways in plants. Research from a team led by Xue-sen Chen at Shandong Agricultural University in China now reveals how the auxin naphthalene acetic acid (NAA) controls production of anthocyanin, the molecule that gives red apples their distinctive coloration. The researchers determined that increasing NAA levels result in reduced production of anthocyanin. These effects are mediated by a subset of auxin-associated proteins that regulate the expression of genes that contribute to the synthesis of this pigment. The color of fruit can strongly influence consumer purchasing decisions, and this signaling pathway could help horticulturalists to ensure that apples retain their visual appeal even under inclement growing conditions.

Enzymatic browning affects the quality of fresh-cut produce. To elucidate the mechanism regulating the browning of red-fleshed apple, we analyzed the transcriptome and metabolome of freshly cut Meihong apple samples collected at 0, 0.5, and 4 h after cutting. The main differentially abundant metabolites during the browning process were phenols, amino acids and fatty acids. Phenols are substrates for enzymatic browning and most of them are negatively correlated with browning. Amino acids and their derivatives are associated with non-enzymatic browning and most of them are positively correlated with browning. A transmission electron microscopy analysis revealed the degradation of the cell membrane of cut fruit samples, which significantly affected the fatty acid content. The differentially expressed genes were mainly related to plant–pathogen interactions. Polyphenol oxidase (PPO) plays a major role during the early browning stage, but PPO gene expression was not higher than WRKY, AP2 and MYB transcription factor.

The soil fungus Fusarium spp, is the main cause of apple replant disease in China. The M9T337 apple rootstock was used to investigate the molecular responses to Fusarium solani infection using transcriptome analysis. We identified 667 differentially expressed genes in F. solani-infected rootstocks, of which 543 were up-regulated and 124 were down-regulated. These differentially expressed genes included receptor kinases, transcription factors, and genes involved in the production and transport of metabolites. In particular, secondary metabolites can enhance plant resistance to pathogens while P450s and UDP-glycosyltransferase play a unique roles in the defense response.

We established a red callus from the leaves of a red-fleshed apple individual, which was a hybrid offspring of the cross between Malus sieversii f. niedzwetzkyana and Malus domestica cv. ‘Fuji’. We analyzed callus growth and anthocyanin biosynthesis/metabolism under different combinations of temperature and light conditions. Incubation in darkness resulted in decreased anthocyanin accumulation, while it promoted callus growth. Exposure to light and low temperature (16 °C) induced the expression of MYB10 and bHLH3/33, which are responsible for coordinating the regulation of anthocyanin biosynthesis, as well as the expression of other structural genes. Treatments with light and high temperature (32 °C) induced MYB16 expression, which repressed anthocyanin biosynthesis. Additionally, low temperature (16 °C) inhibited the expression of MYB111. We analyzed the expression patterns of MYB and bHLH transcription factor genes by quantitative real-time polymerase chain reaction. Our data suggest that light-induced regulation of anthocyanin biosynthesis is primarily caused by altered MYB10 transcript levels, while temperature-induced regulation is the result of changes to the expression of bHLH3/33, MYB16, MYB17, MYB111, and other repressors. In conclusion, we investigated the reciprocal effects of light and temperature on anthocyanin biosynthesis in red-fleshed apple calli. Our findings may provide a theoretical basis for breeding red-fleshed apple varieties with high anthocyanin contents.

Seaweed and its derivatives are widely used as nutrient supplements, biofertilizers, and biostimulants for soil in agriculture. The aim of this study was to evaluate the effects of seaweed fertilizer on the growth of apple (Malus hupehensis Rehd.) seedlings under replant conditions. We investigated the growth of apple seedlings in replant soil treated with seaweed fertilizer at application rates of 0, 5, 20, and 40 g kg−1. The addition of seaweed fertilizer significantly increased the plant height and dry weight of apple seedlings. Seedlings grown in soil treated with seaweed fertilizer, particularly the dose of 40 g kg−1 soil showed higher activities of antioxidant enzymes including superoxide dismutase, peroxidase and catalase, which was accompanied by lower malondialdehyde accumulation. The activities of soil enzymes (invertase, urease, proteinase and phosphatase) were higher in soil treated with seaweed fertilizer than in control soil. An analysis of terminal restriction fragment length polymorphism profiles showed that the fungal communities differed markedly between the 40 g kg−1 seaweed fertilizer treatments and the 0, 5, and 20 g kg−1 treatments. The highest values of Shannon diversity index, evenness index and richness index were in the 40 g kg−1 seaweed fertilizer treatments, and the lowest values of these indexes were in the control. These results suggested that seaweed fertilizer application at a dose of 40 g kg−1 can improve soil enzymes activities, change the soil fungal communities, and improve soil quality. These changes can promote seedlings growth, increase antioxidant activity, and decrease lipid peroxidation in roots, thereby alleviating apple replant disease.

A genetic linkage map of the apple, composed of 175 SSR and 105 SRAP markers, has been constructed using 110 F1 individuals obtained from a cross between the ‘Red Fuji’ Malus domestica and ‘Hongrou’ Malus sieversii cultivars, which have relatively high levels of DNA marker polymorphism and differ remarkably in fruit-related traits. The linkage map comprised 17 linkage groups, covering 1299.67 cM with an average marker distance of 4.6 cM between adjacent markers, or approximately 91% of Malus genome. Linkage groups were well populated and, although marker density ranged from 2.1 to 9.5 cM, just 10 gaps of more than 15 cM were observed. Moreover, just 12.5% of markers displayed segregation distortion. The present genetic linkage map was used to identify quantitative trait loci (QTLs) affecting fruit-related traits. 23 QTLs for ten fruit traits were detected by multiple interval mapping: 3 QTLs for Vc content, One QTL for single fruit weight, 2 QTLs for peel-phenols content, 2 QTLs for flesh-hardness, 2 QTLs for diameter, 6 QTLs for acid content, 1 QTL for sugar content, 2 QTLs for soluble solids content, 2 QTLs for flesh-phenols and 2 QTLs for brittleness. These QTLs were located on linkage groups C1, C2, C3, C5, C6, C7, C9, C10, C14 and C17, respectively. The phenotypic variations exhibited by each QTL ranged from 2% to 72%, and their LOD values varied from 2.03 to 8.93, of which five QTLs were major effect genes (R2 ≥ 10%). The tight linkage markers (*me2em7-460f, *MS01a03-180m, *me1em6-307m, *CH05c06-102f, *me1em8-423f) would be helpful to elucidate the molecular mechanisms of apple domestication and breeding in the future.

Ultraviolet-B (UV-B) radiation promotes anthocyanin synthesis in many plants. Although several transcription factors promote anthocyanin synthesis in response to UV-B radiation, the underlying mechanism remains unclear. In this study, the MdWRKY72 transcription factor gene was isolated from the 'Taishanzaoxia' apple genome. Quantitative real-time PCR analyses revealed that the genes encoding enzymes and transcription factors involved in the anthocyanin synthesis pathway (MdANS, MdDFR, MdUFGT, and MdMYB1) were more highly expressed in MdWRKY72-overexpressing transgenic calli than in the wild-type 'Orin' apple calli. The results indicated that MdWRKY72 increases anthocyanin synthesis in transgenic calli exposed to UV-B radiation. The results of a gel shift assay and chromatin immunoprecipitation proved that MdWRKY72 promotes MdMYB1 expression indirectly by binding to a W-box element in the MdHY5 promoter and directly by binding to a W-box element in the MdMYB1 promoter. Thus, MdWRKY72 increases anthocyanin synthesis via direct and indirect mechanisms. These findings may be useful for elucidating the molecular mechanism underlying UV-B-induced anthocyanin synthesis mediated by MdWRKY72.

Ultraviolet-B (UV-B) radiation and low temperature promote the accumulation of anthocyanins, which give apple skins their red colour. Although many transcription regulators have been characterized in the UV-B and low-temperature pathways, their interregulation and synergistic effects are not well understood. Here, a B-box transcription factor gene, MdBBX20, was characterized in apple and identified to promote anthocyanin biosynthesis under UV-B conditions in field experiments and when overexpressed in transgenic apple calli. The transcript level of MdBBX20 was significantly induced by UV-B. Specific G-box elements in the promoters of target genes were identified as interaction sites for MdBBX20. Further experimental interrogation of the UV-B signalling pathways showed that MdBBX20 could interact with MdHY5 in vitro and in vivo and that this interaction was required to significantly enhance the promoter activity of MdMYB1. MdBBX20 also responded to low temperature (14°C) with the participation of MdbHLH3, which directly bound a low temperature-response cis elements in the MdBBX20 promoter. These findings demonstrate the molecular mechanism by which MdBBX20 integrates low-temperature- and UV-B-induced anthocyanin accumulation in apple skin.

Anthocyanins in apple species are important secondary metabolites that are beneficial for human health. Previous studies revealed that methyl jasmonate (MeJA) promotes anthocyanin accumulation by up-regulating the transcription of related genes. In this study, we isolated a jasmonate (JA)-induced apple MYB gene, MdMYB24-like (MdMYB24L). The encoded nuclear protein contains a conserved R2R3 domain and is homologous to Arabidopsis thaliana AtMYB24. Additionally, MdMYB24L was observed to interact with JA signaling factors (MdJAZ8, MdJAZ11, and MdMYC2) in yeast and in planta. The MdMYC2 protein was also targeted by MdJAZ8 and MdJAZ11, which are rapidly degraded under MeJA treatment. The overexpression of MdMYB24L resulted in higher anthocyanin contents in the transgenic apple 'Orin' calli than in the wild-type control calli. Moreover, the expression levels of the anthocyanin biosynthesis structural genes MdUFGT and MdDFR were up-regulated in the transgenic calli. Furthermore, MdMYB24L positively regulated the transcription of MdDFR and MdUFGT by binding to the MYB-binding site motifs in their promoters. Interestingly, the interaction between MdMYC2 and MdMYB24L further enhanced the transcription of MdUFGT, whereas MdJAZ8 and MdJAZ11 attenuated this effect. We herein provide new details regarding the molecular mechanism by which MYB transcription factors help regulate anthocyanin biosynthesis via JA signaling pathways.