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This study investigated the effects of six crop rotation combinations on the soil quality of old apple orchard and seedling growth of Malus hupehensis Rehd. (apple rootstock) under pot conditions. The inhibitory effects of crops such as Allium fistulosum, Brassica juncea, and Triticum aestivum on four species of Fusarium were observed and compared in six treatments. These were continuous cropping (CK), fumigation with the methyl bromide (FM), rotating A. fistulosum only (R1), rotating A. fistulosum and T. aestivum (R2), rotating A. fistulosum, B. juncea, and T. aestivum (R3), and fallow (FC) in a year. The results showed that the biomass of Malus hupehensis Rehd. seedlings increased significantly. The root length increased and the root architecture was optimized. The respiration rate of the root system was increased by about 1 time after rotation. The treatments of R1, R2, R3, and FC increased bacterial count by 232.17%, 96.04%, 316.21%, and 60.02%, respectively. However, the fungi were reduced in varying degrees and bacteria/fungi ratio was increased by 5–10 times. The enzyme activities, pH, and organic matter were increased, but soil bulk density was decreased. Phenolic acids such as phloridzin was decreased significantly. The copy number of four Fusarium species declined by 85.59%, 74.94%, 69.68%, and 54.41% after rotating three different crops (R3 treatment). The root volatiles of three plants inhibited mycelial growth and spore germination of four Fusarium species.

Apple replant disease (ARD) is a common problem, which occurs in all major apple-growing regions worldwide. It hinders the growth of apple trees and reduces apple yield and quality. Besides, ARD has substantially impeded the development of the apple industry. Fusarium solani, a soil-borne pathogen, is the causative agent of ARD. WRKY transcription factors (TFs) play a crucial role in plant response to biological stress; however, the functional role of apple WRKY-TFs in ARD remains unknown. In this study, F. solani infection apple rootstock ‘M9T337’ induced MdWRKY74 expression was observed. As per the significance analysis, MdWRKY74 expression was positively correlated to endochitinase activity. The MdWRKY74 overexpression in apple callus can significantly improve the resistance to F. solani. In F. solani infection, the relative expression levels of the endochitinase gene (MdECHT) and the activity of endochitinase in the OE-MdWRKY74 calli were higher than those in the control calli. Yeast one-hybrid and electrophoretic mobility transfer analysis revealed the interaction between MdWRKY74 and MdECHT promoter. As per the luciferase reporter analysis, MdWRKY74 significantly increased the activity of the MdECHT promoter. In conclusion, MdWRKY74 was found to be a positive regulator of disease resistance, which improved the apple plant resistance to F. solani infection. This study provides a scientific basis for disease resistance breeding for apple rootstocks.

In this study, the potential regulatory effects of fulvic acid extracted from paper mill effluent (PFA) in apple replant disease (ARD) were investigated through a comprehensive experimental evaluation of the effects of PFA on soil properties, growth inhibition of apple replant pathogens, and growth of replanted Malus hupehensis Rehd. seedlings. PFA with a relatively lower molecular weight was mainly composed of carbohydrates, lignin derivatives, and polysaccharides and was rich in functional groups such as carboxyl and phenolic hydroxyl groups. Treatment with PFA dosages ranging from 2 to 3 g/pot significantly increased available phosphorus (P) in soil by 47.5 to 57.5% when compared with the control without PFA, indicating that PFA had a positive effect in activating P. In addition, PFA stimulated the growth of replanted seedlings by promoting root elongation, enhancing leaf photosynthesis, and increasing the activity of root antioxidant enzymes including superoxide dismutase, peroxidase, and catalase. However, no convincing evidence was found that application of different dosages of PFA had remarkable effects on soil pH, inorganic nitrogen, available potassium, organic matter, and the numbers of bacteria and fungi. Notably, PFA had no effect on the copy number of the main pathogenic fungi causing ARD, including Fusarium oxysporum, Fusarium solani, Fusarium proliferatum, and Fusarium moniliforme. Overall, PFA can alleviate ARD to a certain extent mainly through its effects on improving the resilience of replanted young seedlings rather than by affecting soil microorganisms or providing nutrients.

Apple replant disease (ARD) causes enormous economic loss and threatens the survival of apple industry worldwide. Fusarium solani is one of the pathogens that has been proven to cause ARD. Samples were collected at different time periods to investigate the mechanism of defense responses of apple to F. solani infection by monitoring the biomass, reactive oxygen species (ROS), and antioxidant enzyme activities of the apple rootstock ‘M.9T337’. In addition, the abundance of transcription of four pathogenesis-related (PR) proteins involved in antifungal defense was monitored. The results showed that the apple root system was normal and had small brown areas. However, there is a rapid burst of ROS during the early infection stage, and the activities of antioxidant enzymes and transcription of PRs increased during this period. With the extension in infection time, the infected root tissues displayed dark brown necrosis, and the activities of antioxidant enzymes and abundance of transcription of PRs decreased gradually after reaching their peak. Eventually, the plant biomass decreased, and the plant died. In conclusion, the levels of ROS and activities of antioxidant enzymes played an active role during the early stage of resistance of ‘M.9T337’ apples to infection by F. solani. Infection by F. solani can destroy the ROS scavenging system, causing oxidative damage and inhibiting the growth of apple rootstocks.

CKS (cyclin-dependent kinase subunit) is one of cell cycle genes, and cell cycle in apple (Malus × domestic Borkh.) fruit controlled cell number and fruit size. In order to explore CKS expression during the development of apple fruit size, the cultivar ‘Fuji’ and ‘Ralls’ whose fruit size had obvious difference were used for MdCKS1 gene cloning and expressing. In ‘Fuji’ and ‘Ralls’, the greatest growth rates of fruit size and weight occurred during 7–21 days after full bloom (DAFB), but in ‘Fuji’ during 21–35 DAFB the growth rates of fruit size and weight was still faster, and ‘Fuji’ remained much rapider growth rate of fruit size and weight than ‘Ralls’ within 7–35 DAFB, so the majority of cell production in ‘Ralls’ occurred between 7–21 DAFB and in ‘Fuji’ between 7–35 DAFB, and the biggest difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ occurred at 35 DAFB. MdCKS1 sequence was identical with MdCKS1;1 (CO868566) and apple CKS1 (EB141951). MdCKS1 expression peak in ‘Fuji’ was two-fold higher than in ‘Ralls’; MdCKS1 expression peak in ‘Ralls’ occurred at 21 DAFB and in ‘Fuji’ at 35 DAFB which coincided respectively with rapid growth rate of early fruit. During 140–161 DAFB, ‘Fuji’ fruit had MdCKS1 expression peak again, but ‘Ralls’ not; ‘Fuji’ fruit size and weight had a larger extent of increase than ‘Ralls’, and the second significant difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ occurred at 161 DAFB. In callus, MdCKS1 expression was much higher in ‘Fuji’ than in ‘Ralls’, up-regulated by auxin (NAA) or cytokinin (6-BA) or co-existence of them, and depressed by lower culture temperature. Altogether, ‘Fuji’ fruit had much rapider growth rate than ‘Ralls’ which resulted in significant difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ respectively at 35 DAFB and 161 DAFB, and contributed to their fruit size difference; MdCKS1 expression in ‘Fuji’ fruit was much higher than in ‘Ralls’ both at early stage for fruit cell division and later stage for fruit cell expansion; MdCKS1 expression in callus was up-regulated by auxin or/and cytokinin. Hence, difference of fruit size between ‘Fuji’ and ‘Ralls’ was correlated with growth rate during fruit development, MdCKS1 expression correlated positively with growth rate and cell production of early fruit, and involved in fruit size which maybe was regulated by endogenous auxin or/and cytokine level of genotype.

A pot experiment was performed to investigate the effects of Trichoderma harzianum on the root morphology of Malus hupehensis Rehd. seedlings and their soil environment under replant conditions. The experiment consisted of four treatments: continuously cropped soil (CK1), methyl bromide fumigation (CK2), carrier substrate control (T1), and T. harzianum fertilizer (T2). Plant growth parameters, soil phenolic acid content, abundance of soil microorganisms, and root respiration rate were measured. Compared with CK1, plant height, basal diameter, and fresh weight were 34.58%, 27.55%, and 32.91% greater in T2; 11.35%, 12.10%, and 18.33% greater in T1; and 54.34%, 57.64%, and 45.74% greater in CK2. These metrics were significantly higher in the CK2 treatment than in the other treatments. The second highest values were recorded in the T2 treatment. Differences in root architecture were consistent with differences in biomass. Application of T. harzianum fertilizer was associated with increases of 45.45%, 120.06%, 86.44%, and 268.29% in the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), respectively, and there was little difference between T2 and CK2. The contents of phlorizin and phloretin were 39.39% and 51.70% less in T2, respectively, and 17.85% and 18.14% less in T1, respectively, compared with CK1. Trichoderma harzianum fertilizer increased the abundance of bacteria and actinomycetes while decreasing that of fungi. The gene copy numbers of Fusarium oxysporum and Fusarium moniliforme were 64.30% and 49.35% less, respectively, in the T2 treatment. The fungus population and the gene copy number of Fusarium oxysporum and Fusarium moniliforme was the least in CK2 because of the good sterilization effect. The T. harzianum fertilizer showed satisfactory effects in promoting the root growth of M. hupehensis, increasing the root resistance, decreasing the soil phenolic acid content, and significantly reducing the gene copy number of F. oxysporum and F. moniliforme. In summary, T. harzianum fertilizer is an effective and green alternative for the prevention and control of apple replant disease (ARD).

The effects of different soil chemical amendments (T1, 1‰ quicklime + 1‰ superphosphate; T2, 1‰ quicklime; T3, 1‰ superphosphate) on the soil environment and growth of Malus hupehensis Rehd. seedlings in aged apple orchard soil were studied to provide new insight into the prevention and control of apple replant disease. The amendments differed in their ability to ameliorate the soil environment; nevertheless, they all promoted the growth of M. hupehensis Rehd. seedlings, and the greatest enhancement of growth was observed in T1. On August 15, 2018, soil urease, sucrase, phosphatase, and catalase activities were 1.67 times, 1.32 times, 1.62 times, and 1.35 times higher in T1 compared with CK, respectively. The soil pH increased, which alleviated soil acidification. T1 also promoted the renewal of the community structure and the diversity of soil microorganisms. The copy numbers of Fusarium solani and Fusarium oxysporum were 71.96 and 70.30% lower in T1 compared with CK, respectively. The seedling height and root length of M. hupehensis Rehd. seedlings increased by 40.97 and 289.69% in T1 compared with CK, respectively. Therefore, soil replanting obstacles can be overcome with the application of quicklime and superphosphate; these soil chemical amendments also improve the soil microbial ecological environment and promote the growth of M. hupehensis Rehd. seedlings.

Crop rotation in fruit trees is an effective approach for addressing some of the problems of continuous cropping. To determine whether aged peach orchard soil is suitable for planting apple trees, we studied the effects of two substances abundant in aged peach orchard soil—amygdalin and benzoic acid—on the soil microbial community structure, soil enzyme activity, and the growth of Malus hupehensis Rehd. seedlings. Soils treated with amygdalin (T1), benzoic acid (T2), and a mixed solution of amygdalin and benzoic acid (T3) were used to plant M. hupehensis Rehd. seedlings. Compared with fallow (control) soil, the soil microbial community structure, soil enzyme activities, and root protective enzyme activities, leaf chlorophyll content, and net photosynthetic rate decreased in the three treatments. The biomass and root index of M. hupehensis Rehd. seedlings significantly decreased. Compared with T3, the plant height, ground diameter, fresh weight, dry weight, root length, root surface area, root volume, and root respiration rate of M. hupehensis Rehd. seedlings in T2 in 2015 (2016 in parentheses) decreased by 19.3% (12.6%), 8.7% (7.1%), 21.2% (13.3%), 9.1% (19.6%), 7.9% (25.3%), 40.7% (28.8%), 46.2% (21.1%), and 44.2% (27.5%), respectively. Compared with T3, the same variables in T1 in 2015 (2016 in parentheses) decreased by 34.9% (16.7%), 27.6% (9.8%), 53.6% (19.4%), and 50% (20.5%), 24.1% (31.4%), 55.1% (37.6%), 63.2% (28.2%), and 47.0% (28.7%), respectively. Thus, the inhibitory effect of T3 was the strongest, followed by T2 and T1. In sum, amygdalin and benzoic acid are harmful substances in aged peach orchard soil that inhibit the growth of M. hupehensis Rehd. seedlings.

The relationship between soil texture and the degree of apple replant disease (ARD) was analyzed from the perspective of the microbial community structure and diversity within the rhizosphere soil of Malus hupehensis Rehd. seedlings. Three different textured soils were taken from different apple orchards in Laizhou, Yantai. The soils were divided into two parts, one was kept in replanted conditions, and the other was fumigated with methyl bromide to act as a high standard control. The strength of ARD occurrence was examined by measuring fresh and dry weight suppression (%) of the M. hupehensis seedlings. Differences in the fungal community structure (especially in Fusarium) among the three soil texture types were analyzed using high-throughput sequencing. The results showed that replanted loam clay soil had the highest fungal diversity, followed by sandy loam soil and finally loam soil. The richness of fungi between soil textures, however, was not significantly different. At the genus level, the relative abundance of Fusarium was 1.96%, 0.78%, and 10.89% in replanted sandy loam, replanted loam soil, and replanted loam clay soil, respectively. Moreover, the gene copy number of Fusarium oxysporum, Fusarium solani, and the inhibition rate of fresh weight of M. hupehensis seedlings were the same in the three soil textures. The plant height, photosynthesis (net) (Pn), and stomatal conductance (gS) of the M. hupehensis seedlings were significantly less in the replanted soil compared with the control treatments, with the overall difference being greatest in replanted loam clay soil, followed by replanted sandy loam and then replanted loam soil.

A rapid increase in endogenous ethylene production during ripening is related to fruit softening in the apple (Malus domestica Borkh.) cultivar ‘Taishanzaoxia’. Two genes involved in the ethylene response, MdERF1.1 and MdERF2.1, were isolated and characterized. MdERF1.1 and MdERF2.1 were predicted to encode proteins of 252 and 334 amino acids, respectively. MdERF1.1 and MdERF2.1 were expressed in Escherichia coli, and their molecular weights were determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blotting analyses (28 and 37 kD, respectively). Sequence comparisons and phylogenetic and secondary structure indicated that MdERF1.1 and MdERF2.1 are members of the ERF family. Both of them include a typical GCC‑box binding domain consisting of an α-helix and a three-stranded anti-parallel β-sheet. Sequence comparisons showed that MdERF1.1 is highly identity to MdERF1 from ‘Golden Delicious’, but Gly replaces Ala at position 110 in β-strand 2. This amino acid variation may be involved in fruit softening. Similarly, MdERF2.1 is highly identity to MdERF2 from ‘Golden Delicious’ but differs at three amino acids (Ala, Thr and Glu replace Pro, Ala and Asp at positions 97, 139, and 269, respectively). These three amino acid differences in MdERF2.1 might also be related to fruit softening. In ripening fruit, MdERF1.1 transcript levels rapidly increased during fruit softening in ‘Taishanzaoxia’, and this increase was associated with ethylene production. MdERF2.1 was predominantly expressed in softening ‘Taishanzaoxia’ fruit. The results indicate that ethylene signaling and MdERF1.1 and MdERF2.1 are involved in fruit softening in ‘Taishanzaoxia’.

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...

Apple replant disease (ARD) has been reported in all major fruit-growing regions of the world and is often caused by biotic factors (pathogen fungi) and abiotic factors (phenolic compounds). Soil chemical fumigation can kill soil pathogenic fungi; however, the traditionally used fumigant methyl bromide has been banned because of its ozone-depleting effects. There is thus a need to identify greener fumigant candidates. We characterized the effects of different fumigants on the replanted soil environment and the growth characteristics of Malus hupehensis Rehd. seedlings. All five experimental treatments [treatment 1 (T1), metham-sodium; treatment 2 (T2), dazomet; treatment 3 (T3), calcium cyanamide; treatment 4 (T4), 1,3-dichloropropene; and treatment 5 (T5), methyl bromide] promoted significantly the biomass, root growth, and root respiration rate of M. hupehensis seedlings and the ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3–-N) contents of replanted soil. Metham sodium (T1) and dazomet (T2) had stronger effects compared with 1,3-dichloropropene (T4) and calcium cyanamide (T3). At 172 days after T1, the height, root length, and root respiration rate of Malus hupehensis Rehd. seedlings, and the NH4+-N and NO3–-N contents of replanted soil increased by 91.64%, 97.67%, 69.78%, 81.98%, and 27.44%, respectively, compared with the control. Thus, dazomet and metham sodium were determined to be the optimal fumigants for use in practical applications.

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.

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

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.

Background Light quality significantly affects plant growth and development, photosynthesis, and carbon and nitrogen metabolism. Apple (Malus domestica Borkh.) is a widely cultivated and economically important fruit crop worldwide. However, there are still few studies on the effects of different light qualities on the growth and development of apple seedlings. Results In this study, we explored the effects of blue and red light treatments on the growth and development, photosynthetic characteristics, leaf chloroplast ultrastructure, and carbon and nitrogen metabolism of apple seedlings. Blue light significantly inhibited apple plant growth and leaf extension, but it promoted the development of leaf tissue structures and chloroplasts and positively affected leaf stomatal conductance, the transpiration rate, and photosynthetic efficiency. The red light treatment promoted apple plant growth and root development, but it resulted in loosely organized leaf palisade tissues and low chlorophyll contents. The blue and red light treatments enhanced the accumulation of ammonium nitrogen in apple seedlings. Moreover, the blue light treatment significantly promoted nitrogen metabolism. Additionally, an RNA-seq analysis revealed that both blue light and red light can significantly up-regulate the expression of genes related to carbon and nitrogen metabolism. Blue light can also promote amino acid synthesis and flavonoid metabolism, whereas red light can induce plant hormone signal transduction. The expression of a gene encoding a bHLH transcription factor (MYC2-like) was significantly up-regulated in response to blue light, implying it may be important for blue light-mediated plant development. Conclusions Considered together, blue and red light have important effects on apple growth, carbon and nitrogen metabolism. These findings may be useful for determining the ideal light conditions for apple cultivation to maximize fruit yield and quality.

Background: Apple (Malus domestica Borkh.) is an important economic crop. The pathological effects of Fusarium spp., a group of soilborne pathogens, on the root systems of apple plants was unknown. It was unclear how mycorrhizal apple seedlings resist infection by F. solani. The transcriptional profiles of mycorrhizal and non-mycorrhizal plants infected by F. solani were compared using RNA-Seq.Results: Infection with F. solani significantly reduced the dry weight of apple roots, and the roots of mycorrhizal apple plants were less damaged when the plants were infected with F. solani. They also had enhanced activity of antioxidant enzymes and a reduction in the oxidation of membrane lipids. A total of 1,839 differentially expressed genes (DEGs) were obtained after mycorrhizal and non-mycorrhizal apple plants were infected with F. solani. A gene ontogeny (GO) analysis showed that most DEGs were involved in the binding of ADP and calcium ions. In addition, the enrichment observed from an analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) primarily involved plant-pathogen interaction and Glycolysis/Gluconeogenesis, the mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction. Based on a MapMan analysis, an enormous number of DEGs were involved in the response of mycorrhizal plants to stress. Among them, the overexpressed transcription factor MdWRKY40 significantly improved the resistance of ‘Orin’ to F. solani and the expression of the resistance gene MdGLU by binding the promoter of MdGLU.Conclusion: This paper outlines how the inoculation of apple seedlings by arbuscular mycorrhizal fungi reduces the response to stress at the transcription level of the root system following infection with F. solani, and MdWRKY40 played an important role in the resistance of mycorrhizal apple seedlings to infection with F. solani.

Apple replant disease (ARD) is a common problem in major apple planting areas, and biological factors play a leading role in its etiology. Here, we isolated the bacterial strain QSB-6 from the rhizosphere soil of healthy apple trees in a replanted orchard using the serial dilution method. Strain QSB-6 was provisionally identified as Bacillus amyloliquefaciens based on its morphology, physiological and biochemical characteristics, carbon source utilization, and chemical sensitivity. Maximum likelihood analysis based on four gene sequences [16S ribosomal RNA gene (16S rDNA), DNA gyrase subunit A (gyrA), DNA gyrase subunit B (gyrB), and RNA polymerase subunit B (rpoB)] from QSB-6 and other strains indicated that it had 100% homology with B. amyloliquefaciens, thereby confirming its identification. Flat standoff tests showed that strain QSB-6 had a strong inhibitory effect on Fusarium proliferatum, Fusarium solani, Fusarium verticillioides, Fusarium oxysporum, Alternaria alternata, Aspergillus flavus, Phoma sp., Valsa mali, Rhizoctonia solani, Penicillium brasilianum, and Albifimbria verrucaria, and it had broad-spectrum antibacterial characteristics. Extracellular metabolites from strain QSB-6 showed a strong inhibitory effect on Fusarium hyphal growth and spore germination, causing irregular swelling, atrophy, rupture, and cytoplasmic leakage of fungal hyphae. Analysis of its metabolites showed that 1,2-benzenedicarboxylic acid and benzeneacetic acid, 3- hydroxy-, methyl ester had good inhibitory effects on Fusarium, and increased the length of primary roots and the number of lateral roots of Arabidopsis thaliana plantlet. Pot experiments demonstrated that a QSB-6 bacterial fertilizer treatment (T2) significantly improved the growth of Malus hupehensis Rehd. seedlings. It increased root length, surface area, tips, and forks, respiration rate, protective enzyme activities, and the number of soil bacteria while reducing the number of soil fungi. Fermentation broth from strain QSB-6 effectively prevented root damage from Fusarium. terminal restriction fragment length polymorphism (T-RFLP) and quantitative PCR (qPCR) assays showed that the T2 treatment significantly reduced the abundance of Fusarium in the soil and altered the soil fungal community structure. In summary, B. amyloliquefaciens QSB-6 has a good inhibitory effect on Fusarium in the soil and can significantly promote plant root growth. It has great potential as a biological control agent against ARD.

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.

The MYB transcription factors are important for many aspects of plant stress responses. In this study, we isolated and identified an apple MYB gene, MdMYB108L, whose expression is induced by light and cold stresses. An analysis of MdMYB108L-overexpressing transgenic apple calli revealed that MdMYB108L enhances cold tolerance in apple by upregulating MdCBF3 expression. Interestingly, the expression of MdHY5, which encodes an integrator of light and cold signals, was significantly downregulated in transgenic calli. Yeast one-hybrid and electrophoretic mobility shift assays indicated that MdMYB108L positively regulates cold tolerance by binding to the MdCBF3 promoter. Additionally, MdHY5 functions upstream of MdMYB108L, and the resulting increase in MdMYB108L abundance downregulates MdHY5 transcription. The results of this study elucidate a new pathway for the regulation of apple cold tolerance via a feedback mechanism involving MdMYB108L and MdHY5.

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.

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.

Methyl bromide has been banned worldwide because it causes damage to the ozone layer and the environment. To find a substitute for methyl bromide, the relationships among fumigation, plant growth, and the microbial community in replant soil require further study. We performed pot and field experiments to investigate the effects of dazomet fumigation on soil properties and plant performance. Changes in soil microbial community structure and diversity were assessed using high-throughput sequencing, and plant physiological performance and soil physicochemical properties were also measured. Dazomet fumigation enhanced photosynthesis and promoted plant growth in replant soil; it altered soil physical and chemical properties and reduced soil enzyme activities, although these parameters gradually recovered over time. After dazomet fumigation, the dominant soil phyla changed, microbial diversity decreased significantly, the relative abundance of biocontrol bacteria such as Mortierella increased, and the relative abundance of pathogenic bacteria such as Fusarium decreased. Over the course of the experiment, the soil microbial flora changed dynamically, and soil enzyme activities and other physical and chemical properties also recovered to a certain extent. This result suggested that the effect of dazomet on soil microorganisms was temporary. However, fumigation also led to an increase in some resistant pathogens, such as Trichosporon, that affect soil function and health. Therefore, it is necessary to consider potential negative impacts of dazomet on the soil environment and to perform active environmental risk management in China.

Protoplast transient expression is a powerful strategy for gene functional characterization, especially in biochemical mechanism studies. We herein developed a highly efficient transient expression system for apple protoplasts. The abilities of the Arabidopsis thaliana and Malus domestica ubiquitin-10 (AtUBQ10 and MdUBQ10) promoters to drive the expression of multiple genes were compared with that of the CaMV 35S promoter, and the results revealed that the AtUBQ10 and MdUBQ10 promoters were more efficient in apple protoplasts. With this system, we demonstrated that active AtMKK7ac could activate MAPK6/3/4 signaling cascades, which further regulated MdWRKY33 phosphorylation and stability in apple. Furthermore, the ligand-induced interaction between the immune receptor AtFLS2 and the coreceptor AtBAK1 was reconstituted in apple protoplasts. We also found that the stability of the bacterial effector AvrRpt2 was regulated by feedback involving auxin and the immune regulator RIN4. The system established herein will serve as a useful tool for the molecular and biochemical analyses of apple genes.

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.

Apple (Malus × domestica Borkh.) is one of the most widely produced and economically important fruits in temperate regions. Fruit color development in apples is a major focus for both breeders and researchers as consumers associate brightly colored red apples with ripeness and good flavor. In this chapter, we will discuss research efforts on the coloration of the 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 influencing anthocyanin synthesis, and current goals for breeding for red-skinned and red-fleshed apples. We will also cover key transcription factors, such as MYB, bHLH, and WD40 that are involved in the regulation of anthocyanin synthesis and of fruit color development in apples. We will also discuss the regulation of apple color development by external environmental factors such as light, temperature, and water. Furthermore, we will offer insights into the molecular mechanisms underlying anthocyanin biosynthesis in apples. This knowledge will provide valuable guidance for the breeding of high-quality red-skinned and red-fleshed apple cultivars.

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 Pear is one of the most important fruit crops worldwide. Anthocyanins and procyanidins (PAs) are important secondary metabolites that affect the appearance and nutritive quality of pear. However, few studies have focused on the molecular mechanism underlying anthocyanin and PA accumulation in pear. Results We conducted metabolome and transcriptome analyses to identify candidate genes involved in anthocyanin and PA accumulation in young fruits of the pear cultivar ‘Clapp Favorite’ (CF) and its red mutation cultivar ‘Red Clapp Favorite’ (RCF). Gene–metabolite correlation analyses revealed a ‘core set’ of 20 genes that were strongly correlated with 10 anthocyanin and seven PA metabolites. Of these, PcGSTF12 was confirmed to be involved in anthocyanin and PA accumulation by complementation of the tt19–7 Arabidopsis mutant. Interestingly, PcGSTF12 was found to be responsible for the accumulation of procyanidin A3, but not petunidin 3, 5-diglucoside, opposite to the function of AtGSTs in Arabidopsis. Transformation with PcGSTF12 greatly promoted or repressed genes involved in anthocyanin and PA biosynthesis, regulation, and transport. Electrophoretic mobility shift and luciferase reporter assays confirmed positive regulation of PcGSTF12 by PcMYB114. Conclusion These findings identify a core set of genes for anthocyanin and PA accumulation in pear. Of these, PcGSTF12, was confirmed to be involved in anthocyanin and PA accumulation. Our results also identified an important anthocyanin and PA regulation node comprising two core genes, PcGSTF12 and PcMYB114. These results provide novel insights into anthocyanin and PA accumulation in pear and represent a valuable data set to guide future functional studies and pear breeding.

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.

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

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.

The aim of this experiment was to explore the effects of Allium fistulosum-Brassica juncea-Triticum aestivum rotation on the soil microbial environment and the growth of young apple trees under field conditions. After rotation with A. fistulosum-B. juncea-T. aestivum a year, the β-diversity of soil fungi differed, and there were significant differences in fungal community structure. In addition, cultivable fungi decreased, the ratio of bacteria to fungi increased, and the maximum value was achieved after rotation with B. juncea. The percentage of Fusarium spp. decreased to 1.82%, 1.61%, and 3.32% in Dashaling, Feng Maozhai, and Wantou Village, respectively. The gene copy number of Fusarium oxysporum and Fusarium solani was reduced by 20–50%; the content of phenolic acids, which was mostly phloridzin, decreased significantly by 64.24%, 58.59%, and 71.67% in Dashaling, Feng Maozhai, and Wantou Village, respectively, and the rotation with A. fistulosum resulted in the greatest reduction. The root extracts of A. fistulosum, B. juncea, and T. aestivum inhibited the mycelial growth of four Fusarium species; the root extract of A. fistulosum showed the largest degree of inhibition, followed by B. juncea and T. aestivum. Compared with continuous cropping, the yield was significantly increased under crop rotation, and fruit quality was also enhanced. Thus, A. fistulosum-B. juncea-T. aestivum rotation within a year can promote the growth and yield of young apple trees, reduce the copy number of harmful Fusarium, and changed the structure of the soil microbial community.

Apple replant disease is common in apple cultivation, leading to decay and necrosis of the root tip, growth retardation, and even death of the plant. Biological control, inducing activation of the plant's defense systems, is a promising alternative strategy for controlling the disease. In this study, we used the common rootstock ‘M9T337’ as the test material and harvested the plants at 60 and 120 days after transplantation. We investigated the physiological effects of inoculating the soil with indigenous arbuscular mycorrhizal fungi (AMF) to improve the resistance of the apple rootstock under replant conditions. The Glomus sp. and Paraglomus sp. acted as the major fungal species in QX and LZ. The results showed that inoculation of indigenous AMF increased the biomass of the plants and promoted root growth under replanting conditions. The antioxidant and defense-related enzyme activities in the plants were significantly raised, including the activities of the enzymes superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase. In the seedling root system, the H2O2 content and O2·− production were reduced, resulting in decreased lipid oxidation. Indigenous AMF treatment also induced the activity of chitinase and β-1,3-glucanase. This suggests that inoculation of indigenous AMF enhances the resistance of apple rootstock by regulating root reactive oxygen species levels and the antioxidant system under replanting conditions. This provides a theoretical basis for the prevention and treatment of apple replant disease.

Perennial tree soil inhibits the growth of replanting apples, but the mechanism that underlies this inhibition is poorly understood. A total of 57 perennial tree soils were selected for the collection of soil samples in the Bohai Bay in May 2018. The severity of apple replant disease (ARD) for each soil was determined by calculating the rate of inhibition of growth replanted apple trees. A high-throughput sequencing analysis of internal transcribed spacer (ITS) was used to determine the soil fungal community. A correlation analysis was used to determine the relationship between the rate of inhibition of apple growth and soil factors. The degree of inhibition of plant growth varied substantially among the 57 soil samples examined. Different perennial tree soils have varying degrees of ARD. There was no significant difference in the composition of fungal community at the phylum level, but the genus level differed substantially. The abundances of Fusarium and Mortierella species and the contents of phloridin in the soil and soil organic matter (SOM) were significantly correlated with ARD severity. Structural equation modeling also emphasized that the degree of occurrence of ARD was directly or indirectly affected by Fusarium, Mortierella, phloridin and SOM. A correlation analysis can only be used as an indicator, and further research is merited to reveal how soil parameters affect ARD.