Host-induced gene silencing in wild apple germplasm Malus hupehensis confers resistance to the fungal pathogen Botryosphaeria dothidea.

Host-induced gene silencing (HIGS) is an inherent mechanism of plant resistance to fungal pathogens, resulting from cross-kingdom RNA interference (RNAi) mediated by small RNAs (sRNAs) delivered from plants into invading fungi. Introducing artificial sRNA precursors into crops can trigger HIGS of selected fungal genes, and thus has potential applications in agricultural disease control. To investigate the HIGS of apple (Malus sp.) during the interaction with Botryosphaeria dothidea, the pathogenic fungus causing apple ring rot disease, we evaluated whether apple miRNAs can be transported into and target genes in B. dothidea. Indeed, miR159a from Malus hupehensis, a wild apple germplasm with B. dothidea resistance, silenced the fungal sugar transporter gene BdSTP. The accumulation of miR159a in extracellular vesicles (EVs) of both infected M. hupehensis and invading B. dothidea suggests that this miRNA of the host is transported into the fungus via the EV pathway. Knockout of BdSTP caused defects in fungal growth and proliferation, whereas knockin of a miR159a-insensitive version of BdSTP resulted in increased pathogenicity. Inhibition of miR159a in M. hupehensis substantially enhanced plant sensitivity to B. dothidea, indicating miR159a-mediated HIGS against BdSTP being integral to apple immunity. Introducing artificial sRNA precursors targeting BdSTP and BdALS, an acetolactate synthase gene, into M. hupehensis revealed that double-stranded RNAs were more potent than engineered MIRNAs in triggering HIGS alternative to those natural of apple and inhibiting infection. These results provide preliminary evidence for cross-kingdom RNAi in the apple-B. dothidea interaction and establish HIGS as a potential disease control strategy in apple.

Melatonin promotes the recovery of apple plants after waterlogging by shaping the structure and function of the rhizosphere microbiome.

The dynamics of the physiological adaptability of plants and the rhizosphere soil environment after waterlogging remain unclear. Here we investigated the mechanisms regulating plant condition and shaping of the rhizosphere microbiome in a pot experiment. In the experiment, we added melatonin to waterlogged plants, which promoted waterlogging relief. The treatment significantly enhanced photosynthesis and the antioxidant capacity of apple plants, and significantly promoted nitrogen (N) utilization efficiency by upregulating genes related to N transport and metabolism. Multiperiod soil microbiome analysis showed the dynamic effects of melatonin on the diversity of the microbial community during waterlogging recovery. Random forest and linear regression analyses were used to screen for potential beneficial bacteria (e.g., Azoarcus, Pseudomonas and Nocardioides) specifically regulated by melatonin and revealed a positive correlation with soil nutrient levels and plant growth. Furthermore, metagenomic analyses revealed the regulatory effects of melatonin on genes involved in N cycling in soil. Melatonin positively contributed to the accumulation of plant dry weight by upregulating the expression of nifD and nifK (N fixation). In summary, melatonin positively regulates physiological functions in plants and the structure and function of the microbial community; it promoted the recovery of apple plants after waterlogging stress.

MhCLC-c1, a Cl channel c homolog from Malus hupehensis, alleviates NaCl-induced cell death by inhibiting intracellular Cl- accumulation.

BACKGROUND: Overaccumulation of chloride (Cl) when plants suffer NaCl causes cell damage and death, and is regulated by Cl- channel protein (CLC). Apple roots are very sensitive to Cl-, but information associated with CLC is limited in apple crop that widely cultivated in the world. RESULTS: We identified 9 CLCs from the apple genome and divided them into two subclasses. Among them, MdCLC-c1 promoter contained the largest number of cis-acting elements associated with NaCl stress, and only the MdCLC-c1, MdCLC-d, and MdCLC-g were predicted that may be Cl- antiporters or channels. Expression analysis of MdCLCs homologs in the roots of Malus hupehensis showed that most of the MhCLCs expression were response to NaCl stress, especially MhCLC-c1 expression was upregulated continuously and rapidly expressed during NaCl treatment. Therefore, we isolated MhCLC-c1 and observed it was a plasma membrane-localized protein. The MhCLC-c1 suppression significantly increased sensitivity, reactive oxygen species content, and cell death of apple calli; while MhCLC-c1 overexpression decreased sensitivity, reactive oxygen species content, and cell death of apple calli and Arabidopsis by inhibiting intracellular Cl- accumulation under NaCl stress. CONCLUSIONS: The study selected and isolated a CLC-c gene MhCLC-c1 from Malus hupehensis based on identification of CLCs gene family in apple, and their homologs MhCLCs expression patterns during NaCl treatments, revealing that MhCLC-c1 alleviates NaCl-induced cell death by inhibiting intracellular Cl- accumulation. Our findings confer the comprehensive and in-depth upstanding of the mechanism that plants resist salt stress, and might also confer genetic improvement of salt tolerance in horticultural crops and the development and utilization of saline-alkali land.

Melatonin enhances KCl salinity tolerance by maintaining K+ homeostasis in Malus hupehensis.

Large amounts of potash fertilizer are often applied to apple (Malus domestica) orchards to enhance fruit quality and yields, but this treatment aggravates KCl-based salinity stress. Melatonin (MT) is involved in a variety of abiotic stress responses in plants. However, its role in KCl stress tolerance is still unknown. In the present study, we determined that an appropriate concentration (100 µm) of MT significantly alleviated KCl stress in Malus hupehensis by enhancing K+ efflux out of cells and compartmentalizing K+ in vacuoles. Transcriptome deep-sequencing analysis identified the core transcription factor gene MdWRKY53, whose expression responded to both KCl and MT treatment. Overexpressing MdWRKY53 enhanced KCl tolerance in transgenic apple plants by increasing K+ efflux and K+ compartmentalization. Subsequently, we characterized the transporter genes MdGORK1 and MdNHX2 as downstream targets of MdWRKY53 by ChIP-seq. MdGORK1 localized to the plasma membrane and enhanced K+ efflux to increase KCl tolerance in transgenic apple plants. Moreover, overexpressing MdNHX2 enhanced the KCl tolerance of transgenic apple plants/callus by compartmentalizing K+ into the vacuole. RT-qPCR and LUC activity analyses indicated that MdWRKY53 binds to the promoters of MdGORK1 and MdNHX2 and induces their transcription. Taken together, our findings reveal that the MT-WRKY53-GORK1/NHX2-K+ module regulates K+ homeostasis to enhance KCl stress tolerance in apple. These findings shed light on the molecular mechanism of apple response to KCl-based salinity stress and lay the foundation for the practical application of MT in salt stress.

Bioactive Substances and Biological Functions in Malus hupehensis: A Review.

Malus hupehensis (MH), as a natural resource, contains various active ingredients such as polyphenols, polysaccharides, proteins, amino acids, volatile substances, and other components. Increasingly, studies have indicated that MH showed a variety of biological activities, including antioxidant, hypoglycemic, hypolipidemic, anti-cancer, anti-inflammatory activities, and other activities. Hence, MH has attracted wide interest because of its high medical and nutritional value. It is necessary to review the active components and biological activities of MH. This paper systematically reviewed the chemical substances, biological activities, and potential problems of MH to further promote the related research of MH and provide an important reference for its application and development in medicine and food.

Transcriptome analyses reveal the effects of mixed saline-alkali stress on indoleacetic acid and cytokinins in Malus hupehensis Rehd. leaves.

Saline-alkali stress is a universal abiotic stress factor limiting fruit tree cultivation worldwide. Apple (Malus×domestica Borkh.) is one of the fruits with the largest yields worldwide. Tea crabapple (Malus hupehensis Rehd. var. pingyiensis Jiang) is a type of common apple rootstock in China. Because facultative apomixis occurs in this species, it is often used in molecular research. The present study investigated the molecular mechanism of the response of indoleacetic acid (IAA) and cytokinins [zeatin, trans-zeatin riboside (tZR), isopentenyladenine (iP), and isopentenyladenosine (iPA)] to mixed saline-alkali stress (MSAS) in tea crabapple leaves. The endogenous hormone content of tea crabapple leaves under MSAS was measured, and the expression of stress response-related genes was analyzed by RNA sequencing. The results showed that the concentration of IAA was initially higher and then lower than that in the control, whereas the concentration of zeatin, tZR, iP, and iPA was higher than that in the control. A total of 1262 differentially expressed genes were identified in the three comparison groups. Further analyses suggested that IAA and cytokinin biosynthetic genes were mostly upregulated in tea crabapple leaves, indicating that auxin and cytokinin signaling pathway regulation occurred in response to MSAS. These findings suggest that IAA and cytokinins play an important role in the response of tea crabapple to MSAS. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01275-4.

H2S Enhanced the Tolerance of Malus hupehensis to Alkaline Salt Stress through the Expression of Genes Related to Sulfur-Containing Compounds and the Cell Wall in Roots.

Malus is an economically important plant that is widely cultivated worldwide, but it often encounters saline-alkali stress. The composition of saline-alkali land is a variety of salt and alkali mixed with the formation of alkaline salt. Hydrogen sulfide (H2S) has been reported to have positive effects on plant responses to abiotic stresses. Our previous study showed that H2S pretreatment alleviated the damage caused by alkaline salt stress to Malus hupehensis Rehd. var. pingyiensis Jiang (Pingyi Tiancha, PYTC) roots by regulating Na+/K+ homeostasis and oxidative stress. In this study, transcriptome analysis was used to investigate the overall mechanism through which H2S alleviates alkaline salt stress in PYTC roots. Simultaneously, differentially expressed genes (DEGs) were explored. Transcriptional profiling of the Control-H2S, Control-AS, Control-H2S + AS, and AS-H2S + AS comparison groups identified 1618, 18,652, 16,575, and 4314 DEGs, respectively. Further analysis revealed that H2S could alleviate alkaline salt stress by increasing the energy maintenance capacity and cell wall integrity of M. hupehensis roots and by enhancing the capacity for reactive oxygen species (ROS) metabolism because more upregulated genes involved in ROS metabolism and sulfur-containing compounds were identified in M. hupehensis roots after H2S pretreatment. qRT-PCR analysis of H2S-induced and alkaline salt-response genes showed that these genes were consistent with the RNA-seq analysis results, which indicated that H2S alleviation of alkaline salt stress involves the genes of the cell wall and sulfur-containing compounds in PYTC roots.

Rapid enrichment and separation of two novel minor phenols from Malus hupehensis utilizing liquid-liquid extraction with three-phase solvent system and high-speed counter-current chromatography based on the polarity parameter.

For a rapid enrichment and separation of minor components from Malus hupehensis, the selection of suitable solvent system is the great challenge for liquid-liquid extraction with a three-phase solvent system and high-speed counter-current chromatography. According to the concept of "like dissolves like," the similarity of the average polarity between solvent system and target compounds was the significant characteristic of liquid-liquid extraction with a three-phase solvent system and high-speed counter-current chromatography separation. The polarity parameter model provides a way to calculate the polarity of unknown compounds. Under the guidance of the polarity, an efficient enrichment and separation approach was established through liquid-liquid extraction and high-speed counter-current chromatography with solvent systems composed of n-hexane-ethyl acetate-acetonitrile-water (5:3:5:7, v/v), n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v), respectively. Thus, the total content of minor compounds was increased from 2.6% to 17.2%, and two novel compounds (6´´-O-coumaroyl-2´-O-glucopyranosylphloretin and 3´´´-methoxy-6´´-O-feruloy-2´-glucopyranosylphloretin) were obtained. The discovery of the new dihydrochalcones expanded the structural diversity of compounds produced by the genus Malus. The experimental results demonstrated that compound polarity can be described by the polarity parameter model and is an important reference for investigating optimum solvent systems for liquid-liquid extraction with a three-phase solvent system and high-speed counter-current chromatography.

Ultrasonic-Assisted Aqueous Two-Phase Extraction and Properties of Water-Soluble Polysaccharides from Malus hupehensis.

Malus hupehensis (M. hupehensis), an edible and medicinal plant with significant antioxidant and hypoglycemic activity, has been applied to new resource foods. However, the structural characterization and biological effects of its polysaccharides (MHP) are less known. The optimum extraction parameters to achieve the highest extraction efficiency (47.63%), the yield (1.68%) and purity of MHP (89.6%) by ultrasonic-assisted aqueous two-phase system (ATPS) were obtained under the liquid-to-solid ratio of 23 g/mL, ultrasonic power of 65 W, and ultrasonic time of 33 min. According to the analysis results, MHP was composed of Man, GlcA, Rha, GalA, Glc, Gal, Xyl, Ara, and Fuc, in which Ara and Gal were the main components, and the content of GlcA was the lowest. In in vitro activity analysis, MHP showed a significant antioxidant capacity, and an inhibition activity of α-glucosidase and the advanced glycation end products (AGEs) formation in the BSA/Glc reaction model. MHP interacted with α-glucosidase and changed the internal microenvironment of the enzyme, and inhibited the AGEs formation, which provides more evidence for the antihyperglycemic mechanism of MHP. The results suggest that ATPS is an efficient and environmentally friendly solvent system, and M. hupehensis has broad application prospects in functional foods, healthcare products, and pharmaceuticals.

Optimization, characterization and evaluation of liposomes from Malus hupehensis (Pamp.) Rehd. extracts.

The Malus hupehensis (Pamp.) Rehd. is a traditional medicine and edible plant. The previous study found that the extracts of M. hupehensis (Pamp.) Rehd. had a good antioxidant activity in vivo and in vitro. But its clinical application was limited by its poor solubility, rapidly metabolized and poor bioavailability. Hence, this article aimed at developing liposomes as a novel transdermal system for delivering M. hupehensis extracts efficiently. The prepared liposomes were characterized regarding their entrapment efficiency percentage (EE%), vesicle size (VS), polydispersity index (PDI), zeta potential (ZP) and drug loading (DL). Box-Behnken design response surface methodology and factorial design were used to optimize formulation and preparation process, respectively. The optimized liposomes had an EE of 77.29 ± 0.99%, VS of 102.74 ± 1.61 nm, ZP of -21.79 ± 1.43 mV, PDI of 0.291 ± 0.005 and DL was 6.68 ± 0.49%. Transmission electron microscopy showed liposomes had a regular spherical surface. In addition, liposomes exhibited superior skin permeation potential and retention capacity compared with solution. Histopathological study ensured the safety of liposome application. Meanwhile, the optimized liposome has a good stability. Hence, M. hupehensis extracts liposomes could be considered a promising vehicle for transdermal delivery.

Experimental study on the antioxidant activity of Malus hupehensis (Pamp.) Rehd extracts in vitro and in vivo.

Extracts of Malus hupehensis (Pamp.) Rehder, containing flavonoids with good antioxidant and antiliver injury properties, possess various biological activities. The aim of this study was to explore the antioxidant activity of these extracts in vitro and in vivo. The antioxidant activity of the extracts was studied using scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, and superoxide free radicals and by inhibiting mushroom tyrosinase activity in vitro. An in vivo antioxidant experiment was performed using a rat-aging model. Aging was induced in rats with D-galactose through treating them at doses of extracts about 150, 300, and 600 mg·kg-1 ·day-1 . The Malus hupehensis extracts showed high antioxidant activity; the IC50 values of DPPH radicals, ABTS radicals, superoxide radicals, and mushroom tyrosinase inhibition were 19.00 µg/mL, 303.94 µg/mL, and 3.71 mg/mL, and 1.16 mg/mL, respectively. Our experiments showed that the extracts significantly increased the activity of antioxidant enzymes in the serum and tissue homogenate in vivo, and that the effects were positively correlated with the dose, compared with the activity observed in controls. Histopathological observation also confirmed that the extracts had protective effects after oxidative injury in rat tissues. In conclusion, the extracts of M. hupehensis showed effective antioxidant activity both in vitro and in vivo.

MhMAPK4 from Malus hupehensis Rehd. decreases cell death in tobacco roots by controlling Cd2+ uptake.

Cadmium (Cd) induces cell death in plant roots. Mitogen-activated protein kinase (MAPK) plays a role in the regulation of cell death induced by Cd in plant roots. In this study, MhMAPK4 was isolated from the roots of Malus hupehensis. Subcellular localization showed that the MhMAPK4 protein was located in the cell membrane and cytoplasm and is a transmembrane protein that is characterized by hydrophily. The expression of MhMAPK4 in the roots of M. hupehensis was up-regulated by Cd sulfate and Cd chloride. Phenotypic comparison under Cd stress showed that the growth of wild-type (WT) tobacco was lower than the transgenic lines overexpressing MhMAPK4. The fresh weight and the root length of WT also was lower than that of the transgenic tobacco. The net Cd2+ influx in the tobacco roots was decreased by the overexpression of MhMAPK4, as was root Cd accumulation. The recovery time of the Cd2+ influx to stable state in the transgenic tobacco was also shorter than the WT. The expression of iron-regulated transporter 1 (NtIRT1) and natural resistance associated macrophage protein 5 (NtNRAMP5) was relatively low in the transgenic lines under Cd stress. Cell death and apoptosis in the tobacco roots was reduced following the overexpression of MhMAPK4. The activity of vacuolar processing enzyme (VPE) and the transcript level of VPE in the transgenic tobacco was lower than that of WT under Cd stress. In addition, the electrolyte leakage and malondialdehyde and hydrogen peroxide contents in the transgenic tobacco were lower than those of WT, whereas the antioxidant enzyme activity and expression were higher. These results suggest that MhMAPK4 regulates Cd accumulation by mediating Cd2+ uptake by the roots, and controls Cd-caused cell death by adjusting VPE activity.

Rapid screening and identification of antioxidants in the leaves of Malus hupehensis using off-line two-dimensional HPLC-UV-MS/MS coupled with a 1,1'-diphenyl-2-picrylhydrazyl assay.

The leaves of Malus hupehensis have a strong antioxidant activity and are commonly consumed as a healthy tea. However, detailed information about its antioxidants is incomplete. Herein, we developed an effective strategy based on combining off-line two-dimensional high-performance liquid chromatography with ultraviolet and tandem mass spectrometry detection with a 1,1'-diphenyl-2-picrylhydrazyl assay to rapidly screen and identify the antioxidants from the leaves of M. hupehensis. In the orthogonal two-dimensional liquid chromatography system, a Venusil HILIC column was used for the first dimension, while a Universil XB-C18 column was installed in the second dimension. As a result, 32 antioxidants, including ten dihydrochalcones, two flavanones, nine flavonols, four flavones, and seven phenolic acids were tentatively identified, out of which 23 compounds, as far as we know, were isolated and characterized from the leaves of M. hupehensis for the first time. To the best of our knowledge, this is the first systematic investigation of the antioxidants from the leaves of M. hupehensis. The results indicated that the proposed method is an efficient technique to rapidly investigate antioxidants, especially for coeluted and minor compounds in a complex system.

Complete Chloroplast Genome Sequence of Malus hupehensis: Genome Structure, Comparative Analysis, and Phylogenetic Relationships.

Malus hupehensis belongs to the Malus genus (Rosaceae) and is an indigenous wild crabapple of China. This species has received more and more attention, due to its important medicinal, and excellent ornamental and economical, values. In this study, the whole chloroplast (cp) genome of Malus hupehensis, using a Hiseq X Ten sequencing platform, is reported. The M. hupehensis cp genome is 160,065 bp in size, containing a large single copy region (LSC) of 88,166 bp and a small single copy region (SSC) of 19,193 bp, separated by a pair of inverted repeats (IRs) of 26,353 bp. It contains 112 genes, including 78 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), and four ribosomal RNA genes (rRNAs). The overall nucleotide composition is 36.6% CG. A total of 96 simple sequence repeats (SSRs) were identified, most of them were found to be mononucleotide repeats composed of A/T. In addition, a total of 49 long repeats were identified, including 24 forward repeats, 21 palindromic repeats, and four reverse repeats. Comparisons of the IR boundaries of nine Malus complete chloroplast genomes presented slight variations at IR/SC boundaries regions. A phylogenetic analysis, based on 26 chloroplast genomes using the maximum likelihood (ML) method, indicates that M. hupehensis clustered closer ties with M. baccata, M. micromalus, and M. prunifolia than with M. tschonoskii. The availability of the complete chloroplast genome using genomics methods is reported here and provides reliable genetic information for future exploration on the taxonomy and phylogenetic evolution of the Malus and related species.

Malus hupehensis miR168 Targets to ARGONAUTE1 and Contributes to the Resistance against Botryosphaeria dothidea Infection by Altering Defense Responses.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. MicroRNA168 has been implicated as an essential factor of miRNA pathways by targeting ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC). A fluctuation in AGO1 expression influences various plant-pathogen interactions, and the homeostasis of AGO1 and miR168 accumulation is maintained by a complicated feedback regulatory loop. In this study, the connection between miR168 and the resistance of Malus hupehensis to Botryosphaeria dothidea is revealed. The induction of both the mature miR168 and its precursor in plants subjected to B. dothidea infection indicate the transcriptional activation of MIR168a. MIR168a promoter analysis demonstrates that the promoter can be activated by B. dothidea and salicylic acid (SA). However, the direct target of miR168, M. hupehensis ARGONAUTE1 (MhAGO1), is shown to be induced under the infection. Expression and transcription activity analysis demonstrate the transcriptional activation and the post-transcriptional suppression of MhAGO1 in response to B. dothidea infection. By inhibiting reactive oxygen species (ROS) production and enhancing SA-mediated defense responses, miR168a delays the symptom development of leaves inoculated with B. dothidea and impedes the pathogen growth, while MhAGO1 is found to have the opposite effects. Collectively, these findings suggest that the expression of miR168 and MhAGO1 in M. hupehensis in response to B. dothidea infection is regulated by a complicated mechanism. Targeting to MhAGO1, a negative regulator, miR168 plays a positive role in the resistance by alterations in diverse defense responses.

Exogenous Melatonin Alleviates Alkaline Stress in Malus hupehensis Rehd. by Regulating the Biosynthesis of Polyamines.

Since melatonin was identified in plants decades ago, much attention has been devoted to discovering its role in plant science. There is still a great deal to learn about the functional importance of melatonin, as well as its functional mode. In this paper, we examine the role of melatonin treatment in the response of Malus hupehensis Rehd. to alkaline conditions. Stressed seedlings showed chlorosis and suppressed growth. However, this phenotype was ameliorated when 5 µM melatonin was added to the irrigation solution. This supplementation was also associated with a reduction in cell membrane damage and maintenance of a normal root system architecture. Fewer reactive oxygen species (ROS) were accumulated due to the enhanced scavenging activity of antioxidant enzymes superoxide dismutase, peroxidase, and catalase. In addition, alkaline-stressed seedlings that received the melatonin supplement accumulated more polyamines compared with untreated seedlings. Transcript levels of six genes involved in polyamine synthesis, including SAMDC1, -3, and -4, and SPDS1, -3, and -5, -6, were upregulated in response to melatonin application. All of these results demonstrate that melatonin has a positive function in plant tolerance to alkaline stress because it regulates enzyme activity and the biosynthesis of polyamines.

Dopamine Alleviates Phloridzin Toxicity in Apple by Modifying Rhizosphere Bacterial Community Structure and Function.

Phloridzin significantly influences apple plant growth, development, and resistance to environmental stresses by engaging in various metabolic processes. Its excessive accumulation in soil, attributed to continuous monoculture practices, not only inhibits plant growth but also disrupts the rhizosphere microbial community. This study aims to explore the remedial effects of dopamine, a known antioxidant and stress resistance modulator in plants, on the adverse impacts of phloridzin stress in apple. Through hydroponic and pot experiments, it was demonstrated that dopamine significantly mitigates the growth inhibition caused by phloridzin stress in apple by reducing reactive oxygen species levels and enhancing photosynthesis and nitrogen transport. Additionally, dopamine reduced phloridzin concentrations in both the rhizosphere and roots. Furthermore, dopamine positively influences the structure of the rhizosphere microbial community, enriching beneficial microbes associated with nitrogen cycling. It increases the potential for soil nitrogen degradation and fixation by upregulating the abundance of ureC, GDH, and nifH, as revealed by metagenomic analysis. This aids in alleviating phloridzin stress. The study reveals dopamine's pivotal roles in modulating rhizosphere ecology under phloridzin stress and suggests its potential in sustainable apple cultivation practices to counter ARD and enhance productivity.

Melatonin alleviates cadmium toxicity by regulating root endophytic bacteria community structure and metabolite composition in apple.

The level of cadmium (Cd) accumulation in orchard soils is increasing, and excess Cd will cause serious damage to plants. Melatonin is a potent natural antioxidant and has a potential role in alleviating Cd stress. This study aimed to investigate the effects of exogenous melatonin on a root endophyte bacteria community and metabolite composition under Cd stress. The results showed that melatonin significantly scavenged the reactive oxygen species and restored the photosynthetic system (manifested by the improved photosynthetic parameters, total chlorophyll content and the chlorophyll fluorescence parameters (Fv/Fm)), increased the activity of antioxidant enzymes (the activities of catalase, superoxide dismutase, peroxidase and ascorbate oxidase) and reduced the concentration of Cd in the roots and leaves of apple plants. High-throughput sequencing showed that melatonin increased the endophytic bacterial community richness significantly and changed the community structure under Cd stress. The abundance of some potentially beneficial endophytic bacteria (Ohtaekwangia, Streptomyces, Tabrizicola and Azovibrio) increased significantly, indicating that the plants may absorb potentially beneficial microorganisms to resist Cd stress. The metabolomics results showed that melatonin significantly changed the composition of root metabolites, and the relative abundance of some metabolites decreased, suggesting that melatonin may resist Cd stress by depleting root metabolites. In addition, co-occurrence network analysis indicated that some potentially beneficial endophytes may be influenced by specific metabolites. These results provide a theoretical basis for studying the effects of melatonin on the endophytic bacterial community and metabolic composition in apple plants.

The Mh-miR393a-TIR1 module regulates Alternaria alternata resistance of Malus hupehensis mainly by modulating the auxin signaling.

miRNAs govern gene expression and regulate plant defense. Alternaria alternata is a destructive fungal pathogen that damages apple. The wild apple germplasm Malus hupehensis is highly resistant to leaf spot disease caused by this fungus. Herein, we elucidated the regulatory and functional role of miR393a in apple resistance against A. alternata by targeting Transport Inhibitor Response 1. Mature miR393 accumulation in infected M. hupehensis increased owing to the transcriptional activation of MIR393a, determined to be a positive regulator of A. alternata resistance to either 'Orin' calli or 'Gala' leaves. 5' RLM-RACE and co-transformation assays showed that the target of miR393a was MhTIR1, a gene encoding a putative F-box auxin receptor that compromised apple immunity. RNA-seq analysis of transgenic calli revealed that MhTIR1 upregulated auxin signaling gene transcript levels and influenced phytohormone pathways and plant-pathogen interactions. miR393a compromised the sensitivity of several auxin-signaling genes to A. alternata infection, whereas MhTIR1 had the opposite effect. Using exogenous indole-3-acetic acid or the auxin synthesis inhibitor L-AOPP, we clarified that auxin enhances apple susceptibility to this pathogen. miR393a promotes SA biosynthesis and impedes pathogen-triggered ROS bursts by repressing TIR1-mediated auxin signaling. We uncovered the mechanism underlying the miR393a-TIR1 module, which interferes with apple defense against A. alternata by modulating the auxin signaling pathway.

miR164-NAC21/22 module regulates the resistance of Malus hupehensis against Alternaria alternata by controlling jasmonic acid signaling.

Apple leaf spot disease caused by Alternaria alternata apple pathotype (A. alternata AP) is one of the most severe fungal diseases affecting apple cultivation. Transcription factors are involved in various disease-resistance responses, and many of them are regulated by miRNAs. Here, we performed RNA-Seq to investigate gene expression changes during the defense response of Malus hupehensis against A. alternata AP. NAC21/22 was induced upon A. alternata AP infection and silenced by miR164 via direct mRNA cleavage. Contrasting expression patterns were noted between mature miR164 and NAC21/22 during infection. Contrary to NAC21/22 silencing, transiently overexpressing NAC21/22 in M. hupehensis alleviated disease symptoms on 'gala' leaves, impeded A. alternata AP growth, and promoted jasmonic acid (JA) signaling-related gene expression. Importantly, transient miR164f overexpression in 'gala' leaves enhanced A. alternata AP sensitivity, due perhaps to NAC21/22 downregulation, whereas miR164 suppression produced an opposite effect. In summary, the miR164-NAC21/22 module plays a pivotal role in apple resistance against A. alternata AP by regulating JA signaling.