CovInter: interaction data between coronavirus RNAs and host proteins.

Coronavirus has brought about three massive outbreaks in the past two decades. Each step of its life cycle invariably depends on the interactions among virus and host molecules. The interaction between virus RNA and host protein (IVRHP) is unique compared to other virus-host molecular interactions and represents not only an attempt by viruses to promote their translation/replication, but also the host's endeavor to combat viral pathogenicity. In other words, there is an urgent need to develop a database for providing such IVRHP data. In this study, a new database was therefore constructed to describe the interactions between coronavirus RNAs and host proteins (CovInter). This database is unique in (a) unambiguously characterizing the interactions between virus RNA and host protein, (b) comprehensively providing experimentally validated biological function for hundreds of host proteins key in viral infection and (c) systematically quantifying the differential expression patterns (before and after infection) of these key proteins. Given the devastating and persistent threat of coronaviruses, CovInter is highly expected to fill the gap in the whole process of the 'molecular arms race' between viruses and their hosts, which will then aid in the discovery of new antiviral therapies. It's now free and publicly accessible at: https://idrblab.org/covinter/.

MdMPK3 and MdMPK6 fine-tune MdWRKY17-mediated transcriptional activation of the melatonin biosynthesis gene MdASMT7.

Melatonin (N-acetyl-5-methoxytryptamine) is a potent reactive oxygen species (ROS) scavenger that increases the biotic and abiotic stress tolerance in plants. The signaling and regulation pathways of melatonin in plants remain elusive. Here, we report that transgenic apple (Malus domestica) plants overexpressing the transcription factor gene, MdWRKY17, have higher melatonin contents and lower ROS levels than those of control, while the MdWRKY17 RNA interference (RNAi) lines show the reversed phenotype. The binding of MdWRKY17 to N-acetylserotonin O-methyltransferase7 (MdASMT7) directly promotes the MdASMT7 expression in the in vitro and in vivo. MdASMT7 is a melatonin synthase that localizes to the plasma membrane. MdASMT7 overexpression rescued the lower melatonin contents of MdWRKY17-RNAi lines, confirming the role of MdWRKY17-MdASMT7 module in melatonin biosynthesis in apple. Furthermore, melatonin treatment activated the mitogen-activated kinases (MPKs) MdMPK3 and MdMPK6, which phosphorylate MdWRKY17 to promote transcriptional activation of MdASMT7. RNAi-mediated silencing of MdMPK3/6 decreases MdASMT7 expression in transgenic apple plants overexpressing MdWRKY17, which further confirms MdMPK3/6 fine-tunes MdWRKY17-mediated MdASMT7 transcription. This also forms a positive loop that melatonin activates MdMPK3/6 and thus accelerates the biosynthesis of itself via triggering MdMPK3/6-MdWRKY17-MdASMT7 pathway. This novel melatonin regulatory pathway not only have dissected the molecular mechanisms of melatonin biosynthesis but also provided an alternative approach for generating transgenic melatonin-rich apples which may benefits to human health.

Systematic Description of the Content Variation of Natural Products (NPs): To Prompt the Yield of High-Value NPs and the Discovery of New Therapeutics.

Natural products (NPs) have long been associated with human production and play a key role in the survival of species. Significant variations in NP content may severely affect the "return on investment" of NP-based industries and render ecological systems vulnerable. Thus, it is crucial to construct a platform that relates variations in NP content to their corresponding mechanisms. In this study, a publicly accessible online platform, NPcVar (http://npcvar.idrblab.net/), was developed, which systematically described the variations of NP contents and their corresponding mechanisms. The platform comprises 2201 NPs and 694 biological resources, including plants, bacteria, and fungi, curated using 126 diverse factors with 26,425 records. Each record contains information about the species, NP, and factors involved, as well as NP content data, parts of the plant that produce NPs, the location of the experiment, and reference information. All factors were manually curated and categorized into 42 classes which belong to four mechanisms (molecular regulation, species factor, environmental condition, and combined factor). Additionally, the cross-links of species and NP to well-established databases and the visualization of NP content under various experimental conditions were provided. In conclusion, NPcVar is a valuable resource for understanding the relationship between species, factors, and NP contents and is anticipated to serve as a promising tool for improving the yield of high-value NPs and facilitating the development of new therapeutics.

The MdMEK2-MdMPK6-MdWRKY17 pathway stabilizes chlorophyll levels by directly regulating MdSUFB in apple under drought stress.

Drought stress severely limits plant growth and production in apple (Malus domestica Borkh.). To breed water-deficit-tolerant apple cultivars that maintain high yields under slight or moderate drought stress, it is important to uncover the mechanisms underlying the transcriptional regulation of chlorophyll metabolism in apple. To explore this mechanism, we generated transgenic 'Gala3' apple plants with overexpression or knockdown of MdWRKY17, which encodes a transcription factor whose expression is significantly induced by water deficit. Under moderate drought stress, we observed significantly higher chlorophyll contents and photosynthesis rates in overexpression transgenic plants than in controls, whereas these were dramatically lower in the knockdown lines. MdWRKY17 directly regulates MdSUFB expression, as demonstrated by in vitro and in vivo experiments. MdSUFB, a key component of the sulfur mobilization (SUF) system that assembles Fe-S clusters, is essential for inhibiting chlorophyll degradation and stabilizing electron transport during photosynthesis, leading to higher chlorophyll levels in transgenic apple plants overexpressing MdWRKY17. The activated MdMEK2-MdMPK6 cascade by water-deficit stress fine-tunes the MdWRKY17-MdSUFB pathway by phosphorylating MdWRKY17 under water-deficit stress. This fine-tuning of the MdWRKY17-MdSUFB regulatory pathway is important for balancing plant survival and yield losses (chlorophyll degradation and reduced photosynthesis) under slight or moderate drought stress. The phosphorylation by MdMEK2-MdMPK6 activates the MdWRKY17-MdSUFB pathway at S66 (identified by LC-MS), as demonstrated by in vitro and in vivo experiments. Our findings reveal that the MdMEK2-MdMPK6-MdWRKY17-MdSUFB pathway stabilizes chlorophyll levels under moderate drought stress, which could facilitate the breeding of apple varieties that maintain high yields under drought stress.

MKK4-MPK3-WRKY17-mediated salicylic acid degradation increases susceptibility to Glomerella leaf spot in apple.

Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum fructicola, severely affects apple quality and yield, yet few resistance genes have been identified in apple (Malus domestica Borkh.). Here we found a transcription factor MdWRKY17 significantly induced by C. fructicola infection in the susceptible apple cultivar "Gala." MdWRKY17 overexpressing transgenic "Gala" plants exhibited increased susceptibility to C. fructicola, whereas MdWRKY17 RNA-interference plants showed opposite phenotypes, indicating MdWRKY17 acts as a plant susceptibility factor during C. fructicola infection. Furthermore, MdWRKY17 directly bound to the promoter of the salicylic acid (SA) degradation gene Downy Mildew Resistant 6 (MdDMR6) and promoted its expression, resulting in reduced resistance to C. fructicola. Additionally, Mitogen-activated protein kinase (MAPK) 3 (MdMPK3) directly interacted with and phosphorylated MdWRKY17. Importantly, predicted phosphorylation residues in MdWRKY17 by MAPK kinase 4 (MdMEK4)-MdMPK3 were critical for the activity of MdWRKY17 to regulate MdDMR6 expression. In the six susceptible germplasms, MdWRKY17 levels were significantly higher than the six tolerant germplasms after infection, which corresponded with lower SA content, confirming the critical role of MdWRKY17-mediated SA degradation in GLS tolerance. Our study reveals a rapid regulatory mechanism of MdWRKY17, which is essential for SA degradation and GLS susceptibility, paving the way to generate GLS resistant apple.

Boron nitride nanosheet-assisted matrix solid-phase dispersion microextraction of alkaloids from lotus plumule by high-performance liquid chromatography coupled with ultraviolet detection and ion mobility quadrupole time-of-flight mass spectrometry.

A boron nitride nanosheet (BNNS)-assisted matrix solid-phase dispersion method was established to microextract alkaloids from medicinal plants. The target compounds were identified by high-performance liquid chromatography coupled with ultraviolet detection and ion mobility quadrupole time-of-flight mass spectrometry. During the experimental process, several important parameters, including the type of dispersant, the amount of dispersant, the grinding time, and the type of elution solvent, were optimized. Finally, the BNNSs were chosen as the best dispersant, and their microcosmic morphologies were identified by scanning electron microscopy and transmission electron microscopy. Because of the special property of BNNSs, the cost of this experiment was greatly reduced, especially in elution volume, sample amount (50 mg), and extraction time (2 min). Under the best conditions, 50 mg of sample powder was dispersed with 50 mg of BNNSs, the grinding time was 120 s, the mixed powder was eluted with 200 µL of methanol, and good linearity (r2  > 0.9993) and satisfactory recoveries (80-100%) were obtained. The inter- and intraday precisions were acceptable, with RSDs lower than 2.01 and 4.84%, respectively. The limits of detection ranged from 2.54 to 15.00 ng/mL, and the limits of quantitation were 8.47 to 50.00 ng/mL. The proposed method was successfully applied for the determination of liensinine, isoliensinine, and neferine in lotus plumule.

ELONGATED HYPOCOTYL 5-mediated suppression of melatonin biosynthesis is alleviated by darkness and promotes cotyledon opening.

Melatonin (N-acetyl-5-methoxytryptamine) biosynthesis in plants is induced by darkness and high-intensity light; however, the underlying transcriptional mechanisms and upstream signalling pathways are unknown. We identified a dark-induced and highly expressed melatonin synthetase in Arabidopsis thaliana, AtSNAT6, encoding serotonin N-acetyltransferase. We assessed melatonin content and AtSNAT6 expression in mutants lacking key regulators of light/dark signalling. AtCOP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and AtHY5 (ELONGATED HYPOCOTYL 5), which control light/dark transition and photomorphogenesis, promoted and suppressed melatonin biosynthesis, respectively. Using EMSA and ChIP-qPCR analysis, we showed that AtHY5 inhibits AtSNAT6 expression directly. An analysis of melatonin content in snat6 hy5 double mutant and AtHY5+AtSNAT6-overexpressing plants confirmed the regulatory function of AtHY5 and AtSNAT6 in melatonin biosynthesis. Exogenous melatonin further inhibited cotyledon opening in hy5 mutant and AtSNAT6-overexpressing seedlings, but partially reversed the promotion of cotyledon opening in AtHY5-overexpressing seedlings and snat6. Additionally, CRISPR/Cas9-mediated mutation of AtSNAT6 increased cotyledon opening in hy5 mutant, and overexpression of AtSNAT6 decreased cotyledon opening in AtHY5-overexpressing seedlings via changing melatonin biosynthesis, confirming that AtHY5 decreased melatonin-mediated inhibition of cotyledon opening. Our data provide new insights into the regulation of melatonin biosynthesis and its function in cotyledon opening.

Recent advances on discovery of enzyme inhibitors from natural products using bioactivity screening.

The essence of enzymes is to keep the homeostasis and balance of humans by catalyzing metabolic responses and modulating cells. Suppression of an enzyme slows the progress of some diseases, making it a therapeutic target. Therefore, it is important to develop enzyme inhibitors by proper bioactivity screening strategies for the future treatment of some major diseases. In this review, we summarized the recent (2015-2020) applications of several screening strategies (electrophoretically mediated microanalysis, enzyme immobilization, affinity chromatography, and affinity ultrafiltration) in finding enzyme inhibitors from certain species of bioactive natural compounds of plant origin (flavonoids, alkaloids, phenolic acids, saponins, anthraquinones, coumarins). At the same time, the advantages and disadvantages of each strategy were also discussed, and the future possible development direction in enzyme inhibitor screening has been prospected. To sum up, it is expected to help readers select suitable screening strategies for enzyme inhibitors and provide useful information for the study of the biological effects of specific kinds of natural products.

Apple tree flowering is mediated by low level of melatonin under the regulation of seasonal light signal.

Melatonin regulates the seasonal reproduction in photoperiodic sensitive animals. Its function in plants reproduction has not been extensively studied. In the current study, the effects of melatonin on the apple tree flowering have been systematically investigated. For consecutive 2-year monitoring, it was found that the flowering was always associated with the drop of melatonin level in apple tree. Melatonin application before flowering postponed apple tree flowering with a dose-dependent manner. The increased melatonin levels at a suitable range also resulted in more flowering. The data indicated that similar to the animals, the melatonin also serves as the signal of the environmental light to regulate the plant reproduction. It was mainly the blue and far-red light to regulate the gene expression of melatonin synthetic enzymes and melatonin production in plants. The seasonal alterations of the blue and far-red lights coordinated well with the changes of the melatonin levels and led to decreased melatonin level before flowering. The mechanism studies showed that melatonin per se inhibits all the four flowering pathways in apple. The results not only provide the basic knowledge for melatonin research, but also uncover melatonin as a chemical message of light signal to mediate plant reproduction. This information can be potentially used to control flowering period and prolong the harvest time, helpfully to open a new avenue for increasing crop yield by melatonin application.

[Preparation of hispidulin chitosan microsphere and its antiproliferation effect in vitro].

Hispidulin(HPDL) chitosan microspheres were prepared in this study to deliver HPDL to the lesion sitevia intravenous injection, and further evaluate their anticancer effects in vitro and the growth inhibition effect on A549 cells spheroids. HPDL chitosan microspheres were prepared by emulsion crosslinking method with chitosan as a drug carrier and the amount of HPDL was determined by high performance liquid chromatography (HPLC). The morphology of microspheres was observed under laser scanning confocal microscope. Additionally, the drug release amount of targeting microspheres was detected by dialysis method. Furthermore, the anti-proliferative effects against A549 lung cancer cells were tested by sulforhodamine B (SRB) method, and the effects of HPDL chitosan micrpsphereson early apoptosis of A549 cellswere determined by flow cytometry. A549 cells tumor spheroids were developed in vitro and then HPDL chitosan microspheres were added. On the 0, 1, 3, 7 d after adding the drugs, the inverted microscope was used to observe the mythologicaland volume changes of A549 cells spheroids. The encapsulation efficiency of HPDL chitosan microspheres was （75.32±0.52）%, and the drug loading amount was （7.76±0.67）%. Meanwhile, the microspheres were round shaped andhad smooth surface. The HPDL chitosan microspheres exhibited stronger inhibitory effects on A549 lung cancer cells. The results of flow cytometry indicated that, the early apoptosis rate of lung cancer A549 cells was （37.0±0.75）% at 24 h cells culture after drug administration. The volume of tumor spheroid was significantly inhibited, which had been shrunk by (50.09±11.06)% after the treatment by drug-loaded microsphere at day 7 as compared with blank group; meanwhile, the cells surface were obviously lysed. The preparation method in this research was simple and practicable, and the microspheres prepared with this method were round and smooth, with high encapsulation efficiency, which can significantly inhibit proliferation of lung adenocarcinoma A549 cells and induce cell apoptosis, and at the same time can cause lysisand death of A549 cell tumor spheroid.

The WRKY17-WRKY50 complex modulates anthocyanin biosynthesis to improve drought tolerance in apple.

Drought stress is increasing worldwide due to global warming, which severely reduces apple (Malus domestica) yield. Clarifying the basis of drought tolerance in apple could accelerate the molecular breeding of drought-tolerant cultivars to maintain apple production. We identified a transcription factor MdWRKY50 by yeast two-hybrid (Y2H) assays as an interactor of the drought-tolerant protein MdWRKY17, and confirmed their interaction by bimolecular fluorescence complementation (BiFC) and pull-down assays. MdWRKY50 was induced by drought and when overexpressed in apple, conferred transgenic apple plants enhanced drought tolerance by directly binding to the promoter of anthocyanin synthetic gene Chalcone synthase (MdCHS) to upregulate its expression for higher anthocyanin. Increased anthocyanin relieves apple plants from oxidative damage under drought stress. MdWRKY50 RNA-interference transgenic apple plants showed opposite phenotypes. The dimerization of MdWRKY50 with mutated MdWRKY17DP mimicking drought-induced phosphorylation by the mitogen-activated protein kinase kinase 2 (MEK2)-MPK6 cascade, compared with MdWRKY17AP and MdWRKY17, further promoted anthocyanin biosynthesis, suggesting dimerization with MdWRKY17 makes MdWRKY50 more powerful in promoting anthocyanin biosynthesis under drought stress. Taken together, we isolated an entire MEK2-MAPK6-MdWRKY17-MdWRKY50-MdCHS pathway for drought tolerance and generated transgenic apple germplasm with enhanced drought tolerance and higher anthocyanin levels.

A systematic review of state-of-the-art strategies for machine learning-based protein function prediction.

New drug discovery is inseparable from the discovery of drug targets, and the vast majority of the known targets are proteins. At the same time, proteins are essential structural and functional elements of living cells necessary for the maintenance of all forms of life. Therefore, protein functions have become the focus of many pharmacological and biological studies. Traditional experimental techniques are no longer adequate for rapidly growing annotation of protein sequences, and approaches to protein function prediction using computational methods have emerged and flourished. A significant trend has been to use machine learning to achieve this goal. In this review, approaches to protein function prediction based on the sequence, structure, protein-protein interaction (PPI) networks, and fusion of multi-information sources are discussed. The current status of research on protein function prediction using machine learning is considered, and existing challenges and prominent breakthroughs are discussed to provide ideas and methods for future studies.

A systematic review on the state-of-the-art strategies for protein representation.

The study of drug-target protein interaction is a key step in drug research. In recent years, machine learning techniques have become attractive for research, including drug research, due to their automated nature, predictive power, and expected efficiency. Protein representation is a key step in the study of drug-target protein interaction by machine learning, which plays a fundamental role in the ultimate accomplishment of accurate research. With the progress of machine learning, protein representation methods have gradually attracted attention and have consequently developed rapidly. Therefore, in this review, we systematically classify current protein representation methods, comprehensively review them, and discuss the latest advances of interest. According to the information extraction methods and information sources, these representation methods are generally divided into structure and sequence-based representation methods. Each primary class can be further divided into specific subcategories. As for the particular representation methods involve both traditional and the latest approaches. This review contains a comprehensive assessment of the various methods which researchers can use as a reference for their specific protein-related research requirements, including drug research.

Analysis of iodinated amino acids by microemulsion electrokinetic chromatography using aliphatic amine as a cosurfactant.

A novel microemulsion electrokinetic chromatography (MEEKC) was established for the separation and determination of iodinated amino acids using n-butylamine as a novel cosurfactant. By optimizing the type of oil phase, the type and concentration of surfactant, the concentration of cosurfactant and the type and concentration of buffer in the microemulsion system, the optimal conditions for the separation of organic iodines were determined to be 0.5% ethyl acetate, 0.6% SDS, 1.2% n-butylamine and 10 mM sodium borate. The efficient and rapid separation of the five analytes (3-iodo-L-tyrosine (MIT), 3, 5-Diiodo-L-tyrosine (DIT), 3, 5-Diiodo-L-thyronine (T2), 3, 3', 5-Triiodo-L-thyronine (T3) and L-Thyroxine (T4)) was achieved under the optimal conditions. The reliability of the method was verified by measuring the precision, LOD, LOQ and recovery. The practicality of the MEEKC method was demonstrated by applying it to the determination of two iodotyrosines, MIT and DIT, in kelp. The analytical method established in this experiment will provide a reference for the study of iodotyrosines in other natural plants and food.

Ion pair-based mobile phase additives to improve the separation of alkaloids in supercritical fluid chromatography.

In this study, a supercritical fluid chromatography (SFC) method based on ion pair reagents was used to separate alkaloids. The chromatographic parameters, including the stationary phase, additive type, additive concentration, outlet pressure, temperature and flow rate, were optimized. Baseline separation was completed in 20 min on an Agilent Pursuit 5 PFP column (4.6 × 150 mm) using carbon dioxide as the mobile phase and 7.5 mM sodium 1-pentanesulfonate as an additive with gradient elution at 140 bar, 60 °C, and a flow rate of 1.5 mL/min. The retention rate and resolution of the analytes were satisfactory. The limits of detection were 27.04-298.03 ng/mL, and the limits of quantification were 90.15-993.42 ng/mL. The recoveries of low and high concentrations were 77.46-111.86% and 83.84-111.00%, respectively. This ion pair additive greatly improved the separation efficiency of alkaloids. Consequently, this SFC method was successfully applied to the separation of alkaloids from Rhizoma corydalis.

Phase â and phase â ¡ metabolic studies of Citrus flavonoids based on electrochemical simulation and in vitro methods by EC-Q-TOF/MS and HPLC-Q-TOF/MS.

The oxidation products and metabolic pathways of five Citrus flavonoids were studied by online electrochemical/quadrupole time-of-flight mass spectrometry (EC/Q-TOF/MS). The simulated oxidation metabolism of target compounds in phase I and phase Ⅱ was carried out at boron-doped diamond (BDD) working electrode. The results obtained by EC-MS were compared with the conventional metabolism of rats and humans reported in previous literatures. In addition, the method of incubating the target compounds with rat liver microsomes in vitro was established, the target compounds and their metabolites were analyzed by high performance liquid chromatography coupled mass spectrometry. The structures of the metabolites were determined by accurate mass measurements and previous in vivo metabolite results. The results showed that the electrochemical oxidation metabolites were consistent with the results of in vitro incubation of liver microsomes, and also with the results reported in other literatures. As a consequence, EC/Q-TOF/MS is a promising and effective tool for studying metabolic transformation of different complex food components.

Potential roles of melatonin and ABA on apple dwarfing in semi-arid area of Xinjiang China.

Dwarfing is a typic breeding trait for mechanical strengthening and relatively high yield in modern apple orchards. Clarification of the mechanisms associated with dwarfing is important for use of molecular technology to breed apple. Herein, we identified four dwarfing apple germplasms in semi-arid area of Xinjiang, China. The internodal distance of these four germplasms were significantly shorter than non-dwarfing control. Their high melatonin (MT) contents are negatively associated with their malondialdehyde (MDA) levels and oxidative damage. In addition, among the detected hormones including auxin (IAA), gibberellin (GA), brassinolide (BR), zeatin-riboside (ZR), and abscisic acid (ABA), only ABA and ZR levels were in good correlation with the dwarfing phenotype. The qPCR results showed that the expression of melatonin synthetic enzyme genes MdASMT1 and MdSNAT5, ABA synthetic enzyme gene MdAAO3 and degradative gene MdCYP707A, ZR synthetic enzyme gene MdIPT5 all correlated well with the enhanced levels of MT, ABA and the reduced level of of ZR in the dwarfing germplasms. Furthermore, the significantly higher expression of ABA marker genes (MdRD22 and MdRD29) and the lower expression of ZR marker genes (MdRR1 and MdRR2) in all the four dwarf germplasms were consistent with the ABA and ZR levels. Considering the yearly long-term drought occurring in Xinjiang, China, it seems that dwarfing with high contents of MT and ABA may be a good strategy for these germplasms to survive against drought stress. This trait of dwarfing may also benefit apple production and breeding in this semi-arid area.

Carbonized biosorbent assisted matrix solid-phase dispersion microextraction for active compounds from functional food.

In this study, mangosteen peel based activated carbon was prepared and first applied as adsorbent in matrix solid-phase dispersion (MSPD) for simultaneously extraction of flavonoids from Dendrobium huoshanense prior to their separation and determination by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS). The MSPD-UHPLC-Q-TOF/MS method was validated exhaustively. Good linearities (r2 ≥ 0.9929) were obtained for all target analytes. The limits of detection was in the range of 0.00387-0.159 µg/g. Satisfactory recoveries of six target compounds were between 80.02 and 99.49% and 85.32-99.86% for the low and high spiked level, respectively. Furthermore, relative to other common sorbent, the prepared mangosteen peel based activated carbon was less expensive and more environmentally-friendly. Consequently, the proposed method was a simple, efficient, low-cost, eco-friendly, time-saving and sensitive approach that could be successfully applied to the extraction and determination of flavonoids compounds in complex matrix.

MdWRKY11 improves copper tolerance by directly promoting the expression of the copper transporter gene MdHMA5.

Overuse of fungicides and fertilizers has resulted in copper (Cu) contamination of soils and toxic levels of Cu in apple fruits. To breed Cu-resistant apple (Malus domestica) cultivars, the underlying molecular mechanisms and key genes involved in Cu resistance must be identified. Here, we show that MdWRKY11 increases Cu tolerance by directly promoting the transcription of MdHMA5. MdHMA5 is a Cu transporter that may function in the storage of excess Cu in root cell walls and stems for Cu tolerance in apple. The transcription factor MdWRKY11 is highly induced by excess Cu. MdWRKY11 overexpression in transgenic apple enhanced Cu tolerance and decreased Cu accumulation. Apple calli transformed with an MdWRKY11-RNAi construct exhibited the opposite phenotype. Both an in vivo chromatin immunoprecipitation assay and an in vitro electrophoretic mobility shift assay indicated that MdWRKY11 binds to the promoter of MdHMA5. Furthermore, MdWRKY11 promoted MdHMA5 expression in transgenic apple plants, as revealed by quantitative PCR. Moreover, inhibition of MdWRKY11 expression by RNA interference led to a significant decrease in MdHMA5 transcription. Thus, MdWRKY11 directly regulates MdHMA5 transcription. Our work resulted in the identification of a novel MdWRKY11-MdHMA5 pathway that mediates Cu resistance in apple.