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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.

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.

The transformation of sugars is a sustainable method of production of high value-added chemicals and fuels. Traditional procedures used H2SO4 for sugar dehydration, followed by hydrogenation with f...

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.

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.

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.

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.

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.

The integration of thermoelectric (TE) device on fabrics is a challenge. Instead of using adhesive tape to fix traditional filmlike TE devices onto the fabrics, which sacrifices the breathability of fabrics, here we report a flexible TE fiber based on poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS)/tellurium nanowires (Te NWs) composites, which can be woven and integrated into the fabric directly. The TE fibers have a tunable content and orientation of Te NWs and also the potential to be scaled up based on the wet-spinning process. The TE fibers exhibit desirable wearable TE performances, including high power factor (78.1 μW m-1 K-2), high mass-specific power (9.48 μW g-1), excellent mechanical flexibility, and superior integrability.

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.

Microstructure, hardness and fatigue properties of X30N high nitrogen stainless bearing steel were investigated. It was found that nitrogen addition could effectively reduce the amount and size of coarse carbides. The original austenite grain size was obviously refined. Additionally, more retained austenite was found in X30N steel after quenching at 1050 °C, which could be reduced from about 30% to about 6% by cold treatment at − 73 °C and subsequent tempering, and thus, the ultimate hardness was increased up to about 61 HRC with reduction of austenite and precipitation of carbonitrides. Furthermore, the rolling contact fatigue lives of X30N steel are superior to those of 440C steel, which was attributed to the enhanced hardness and a certain retained austenite in the high nitrogen steel.

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...

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.

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.

Microstructure and property of bearing steel with and without nitrogen addition were investigated by microstructural observation and hardness measurement after different heat treatment processing. Based on the microstructural observation of both 9Cr18 steel and X90N steel, it was found that nitrogen addition could effectively reduce the amount and size of coarse carbides and also refine the original austenite grain size. Due to addition of nitrogen, more austenite phase was found in X90N steel than in 9Cr18 steel. The retained austenite of X90N steel after quenching at 1050 °C could be reduced from about 60% to about 7% by cold treatment at −73 °C and subsequent tempering, and thus finally increased the hardness up to 60 HRC after low temperature tempering and to 63 HRC after high temperature tempering. Furthermore, both the wear and corrosion resistance of X90N steel were found much more superior than those of 9Cr18 steel, which was attributed to the addition of nitrogen. It was proposed at last that nitrogen alloying into the high chromium bearing steel was a promising way not only to refine the size of both carbides and austenite, but also to achieve high hardness, high wear property and improved corrosion resistance of the stainless bearing steel.

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.

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.

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.

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.

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.

Anthocyanin and proanthocyanidin (PA) play important roles in plant growth and development. Although previous studies have identified many of the transcription factors involved in the anthocyanin and PA pathway, the regulation mechanisms of these pathways remain poorly understood. In this study, we identified a NAC transcription factor, MdNAC52, whose gene transcript levels increased during apple coloration. Apple calli overexpressing MdNAC52 accumulated anthocyanin. Yeast one-hybrid, electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that MdNAC52 could interact with the promoters of MdMYB9 and MdMYB11 to regulate anthocyanin biosynthesis. MdNAC52 was targeted by MdHY5 in response to light. Interestingly, MdNAC52 participated in the regulation of PA biosynthesis through controlling the expression of MdMYB9 and MdMYB11. MdNAC52 could also bind to the LAR promoter to regulate its expression and promote PA synthesis. Overall, these findings establish that MdNAC52 binds to the promoters of MdMYB9 and MdMYB11 to promote anthocyanin and PA biosynthesis and directly regulates LAR to modulate PA metabolism. Our study provides new insights into the roles of a NAC transcription factor in regulating anthocyanin and PA accumulation in apple.

The regulator MdERF1B in the apple (Malus × domestica) ethylene pathway mainly acts on MdMYB9 and MdMYB11 to regulate anthocyanin and proanthocyanidin accumulation. Dietary anthocyanins and proanthocyanidins (PAs) have health benefits for humans, and are associated with decreased risks of coronary heart disease and cancer. Ethylene can enhance reddening of apple (Malus × domestica), but the regulatory mechanism is poorly understood. In this study, an ethylene response factor (ERF), MdERF1B, was identified and functionally characterized. ‘Orin’ calli overexpressing MdERF1B were generated and then analyzed by quantitative reverse transcription-PCR. Compared with the control calli, the MdERF1B-overexpressing calli showed increased expression levels of MdACO1, MdERF1, and MdERF3 in the ethylene pathway and MdCHS, MdCHI, MdF3H, MdDFR, MdANS, MdLAR, MdANR, MdMYB9 and MdMYB11 in the flavonoid pathway. As a result, the levels of anthocyanins and PAs were significantly increased in the MdERF1B-overexpressing calli. MdERF1B interacted with MdMYB9, MdMYB1, and MdMYB11 proteins in yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays. Furthermore, in yeast one-hybrid and electrophoretic mobility shift assays, MdERF1B also bound to the promoters of MdMYB9, MdMYB1, and MdMYB11. In a luciferase reporter assay, MdERF1B mainly activated proMdMYB9 and proMdMYB11, promoting their expression levels. This was in agreement with MdERF1B’s overexpression in calli, which barely affected MdMYB1 expression. Taken together, our findings provide an insight into the regulatory mechanisms in the ethylene pathway that increase anthocyanin and PA accumulation in apple.

The cold-induced metabolic pathway and anthocyanin biosynthesis play important roles in plant growth. In this study, we identified a bHLH binding motif in the MdMYB15L protein using protein sequence analyses. Yeast two-hybrid and pull-down assays showed that MdMYB15L could interact with MdbHLH33. Overexpressing MdMYB15L in red-fleshed callus inhibited the expression of MdCBF2 and resulted in reduced cold tolerance but did not affect anthocyanin levels. Chip-PCR and EMSA analysis showed that MdMYB15L could bind the type II cis-acting element found in the MdCBF2 promoter. Overexpressing MdMYB15L in red-fleshed callus overexpressing MdbHLH33 also reduced cold tolerance and reduced MdbHLH33-induced anthocyanin biosynthesis. Knocking out the bHLH binding sequence of MdMYB15L (LBSMdMYB15L) prevented LBSMdMYB15L from interacting with MdbHLH33. Overexpressing LBSMdMYB15L in red-fleshed callus overexpressing MdbHLH33 also reduced cold tolerance and reduced MdbHLH33-induced anthocyanin biosynthesis. Together, these results suggested that an apple repressor MdMYB15L might play a key role in the cold signaling and anthocyanin metabolic pathways.

In this paper, a novel collector, 2-ethyl-2-hexenoic hydroxamic acid (EHHA) was prepared and characterized by elemental analysis, infrared, 1H NMR, 13C NMR and mass spectra. The flotation performance and adsorption mechanism of EHHA to ilmenite were investigated by micro-flotation tests, density functional theory (DFT) calculations, FTIR spectra, zeta potential and solution chemistry analyses. The micro-flotation results indicated that EHHA exhibited superior flotation performance compared to isooctyl hydroximic acid (IOHA) and octyl hydroxamic acid (OHA), and floated out 84.03% ilmenite at pH 8.0 with 250 mg/L dosage. The analyses of FTIR spectra and zeta potential demonstrated that EHHA might chemisorb onto ilmenite surfaces by form of five-membered chelates. The solution chemistry analyses further inferred that at pH 6.3–10.5, both Fe and Ti species on ilmenite surfaces could chelate EHHA. DFT calculation results implied EHHA owned the strongest affinity to ilmenite among the three C8 hydroximic acids. To discern the sharply improving floatability of ilmenite at pH 8–10, a schematic co-adsorption molecule–ion model of EHHA on ilmenite surfaces was suggested.

One-pot synthesis of cyclic aldol tetramer and α, β-unsaturated aldol from C3-C8 linear aldehydes using phase-transfer catalyst (PTC), quaternary ammonium, combined with sodium hydroxide as catalysts was investigated. Butanal was subjected for detail investigations to study the effect of parameters. It was found that the selectivity of cyclic aldol tetramer depends greatly on the operating conditions of the reaction, especially the PTC/butanal molar ratio. The average selectivity of 2-hydroxy-6-propyl-l, 3, 5-triethyl-3-cyclohexene-1-carboxaldehyde (HPTECHCA) was 54.41% using tetrabutylammonium chloride combined with 14% (mass fraction) NaOH as catalysts at 60 °C for 2 h with a PTC-to-butanal molar ratio of 0.09:1. Pentanal was more likely to generate cyclic aldol tetramer compared with other aldehydes under the optimum experimental conditions. Recovery of the PTC through water washing followed by adding enough sodium hydroxide from the washings was also demonstrated.

The flotation behavior and adsorption mechanism of α-hydroxyoctyl phosphinic acid (HPA) to malachite were investigated by micro-flotation tests, zeta potential measurements, and FTIR and XPS analysis. The micro-flotation results demonstrated that compared with sodium isobutyl xanthate (SIBX) and styrene phosphonic acid (SPA), HPA exhibited superior collecting performances to direct flotation recovery of malachite and floated out above 80% malachite at pH 5–10. The results of zeta potential measurement inferred that there might be covalent bonds between HPA and copper on the malachite surface. The FTIR analysis confirm that HPA might react with copper species of malachite surfaces to form Cu–HPA complex adsorption layers, with a release of H+ ions (from P H group). The XPS analysis provided very clear evidence that the Cu(II) species was partly reduced to Cu (I) species during HPA chemisorption on malachite surfaces, accompanying P(III) oxidation to P(V) species of HPA.

A novel collector 2-ethyl-2-hexenal oxime was synthesized from 2-ethyl-2-hexenal and hydroxylamine hydrochloride. The yield of oxime was 78.05% under the optimum experimental conditions. The oxime was characterized by elemental analysis, infrared spectrum, mass spectrum, 1 H NMR and 13 C NMR spectroscopy. The results of ultraviolet spectrum showed that the oxime exhibited stronger reaction affinity with Cu 2+ than with Ni 2+ or Fe 3+ , etc. Infrared spectroscopic analysis implicated that the oxime bound to Cu 2+ through both the C C and C N groups. Microflotation tests of pure malachite mineral and chalcopyrite mineral indicated that the recovery of chalcopyrite was 91.16% under the flotation condition of rougher pH 10 and collector concentration of 200 mg/L, and the recovery of malachite reached 90.56% under the flotation condition of rougher pH 11.5 and collector concentration of 250 mg/L. The results of flotation tests of sulfide–oxide copper from Dexing copper mine showed that 2-ethyl-2-hexenal oxime achieved an excellent mean concentration containing 4.52% Cu with 79.54% Cu recovery, and the Cu average recovery increased by 2.35% compared to that of butyl xanthate.

Anthocyanin biosynthesis is induced by cytokinins, and is regulated by MYB transcription factors. However, the underlying molecular mechanisms have not been fully characterised. In the present study, red-fleshed apple callus were induced from the leaves of an R6/R6 homozygous line, which was the hybrid offspring of Malus sieversii f. niedzwetzkyana and ‘Fuji’. We analysed the callus anthocyanin contents in response to different cytokinin concentrations. We observed that cytokinin treatments upregulated the expression of anthocyanin structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3. Additionally, the expression of MdMYBL2, which encodes the bHLH and EAR motifs, was inhibited by cytokinin treatments. The MdMYBL2-overexpressing callus had lower anthocyanin contents than the wild-type controls. We noted that the expression levels of anthocyanin biosynthesis structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3 were strongly suppressed in the transgenic callus. Subsequent yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MdMYBL2 interacts with MdbHLH3, which may influence the expression of anthocyanin biosynthesis-related genes. Our findings may provide new insights into how MYB transcription factors influence the cytokinin-regulated anthocyanin biosynthesis in red-fleshed apples.

Protein-based nanoparticles represent a promising approach to carry polypeptide and protein drugs. Using both theory and experimentation, an interferon α-1b (IFN) delivery system carried by bovine serum albumin (BSA) nanoparticles was designed. Theoretical results indicate the most probable binding site and interaction mechanism for IFN on BSA. IFN has a higher binding affinity with BSA compared with small chemical drugs. The drug loading is about 8 mg/g, significantly higher than those reported in other literature. The release profiles differ between the nanoparticles prepared by the incorporation method and the adsorption method. The adsorption of IFN on BSA nanoparticles is monolayer adsorption. The fact that IFN was carried successfully by BSA nanoparticles establishes a solid basis for expanding the drug loading field of BSA nanoparticles to proteins and polypeptides.