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Apple (Malus × domestica Borkh.) is a perennial woody plant that often suffers from various biological stresses. Many harmful pathogens can infect apple trees and lead to reduced production. We comprehensively identified the WRKY genes in the apple genome and analyzed their expression in response to several biological stressors, including Alternaria alternata, Pythium ultimum, Botryosphaeria dothidea, Erwinia amylovora, Penicillium expansum, Gymnosporangium yamadae, and Apple replant disease. There were 113 MdWRKYs identified in the apple genome. Twenty-two MdWRKYs were differentially expressed in response to at least five pathogens. Promoter sequence analysis showed that these genes carried many defense- and stress-responsive elements, such as MeJA-response elements, salicylic acid-response elements, and W-box elements, in their promoters. Transient expression assays showed that MdWRKY40a and MdWRKY54h played negative roles in defense against B. dothidea infection. WRKY40 and WRKY60 and the MdWKRY33s might play important roles in responding to pathogens and are conserved in some plants. These differentially expressed MdWRKYs might play key roles in the apple response to multiple pathogens.

Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

Mango (2n = 2x = 40) is an important tropical/subtropical evergreen fruit tree grown worldwide and yields nutritionally rich and high-value fruits. Here, a high-quality mango genome (396 Mb, contig N50 = 1.03 Mb) was assembled using the cultivar “Irwin” from Florida, USA. A total of 97.19% of the sequences were anchored to 20 chromosomes, including 36,756 protein-coding genes. We compared the β-carotene content, in two different cultivars (“Irwin” and “Baixiangya”) and growth periods. The variation in β-carotene content mainly affected fruit flesh color. Additionally, transcriptome analysis identified genes related to β-carotene biosynthesis. MiPSY1 was proved to be a key gene regulating β-carotene biosynthesis. Weighted gene co-expression network analysis, dual luciferase, and yeast one-hybrid assays confirmed that transcription factors (TFs) MibZIP66 and MibHLH45 activate MiPSY1 transcription by directly binding to the CACGTG motif of the MiPSY1 promoter. However, the two TFs showed no significant synergistic effect on promoter activity. The results of the current study provide a genomic platform for studying the molecular basis of the flesh color of mango fruit.

Apple is one of the most important fruit trees in temperate zones, and is cultivated widely throughout the world. Drought stress affects the normal growth of apple tree, and further affects fruit yield and quality. The present study examined the effects of drought on photosynthesis and water use efficiency (WUE) of two apple cultivars (Honeycrisp and Yanfu 3) that differ in drought tolerance. The results showed that the photosynthetic rate decreased in response to drought stress for both cultivars, with significant differences in intensity. Values for net photosynthetic rate (Pn) in stressed Yanfu 3 remained significantly lower than in the controls, while, for Honeycrisp, only a slight drop in photosynthesis. Similarly, stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr) were markedly reduced in Yanfu 3 under drought stress. However, Honeycrisp showed only minor changes. Under drought stress, the contents of Chl a, Chl b and Chl t in Yanfu 3 were all decreased significantly compared with the control. However, little difference in Honeycrisp was noted between stressed plants and controls. Values for WUE in stressed Yanfu 3 remained higher than in the controls from day 3 until the end of the experiment, while no significant difference was observed in Honeycrisp. Furthermore, Honeycrisp also exhibited superior physiological traits, as indicated by its anatomical and morphological characteristics. Therefore, we conclude that the superior drought tolerance of Honeycrisp was due to its anatomical and morphological characteristics, which possibly contributed to the maintenance of higher photosynthetic capacity than Yanfu 3.

Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple 'Golden Delicious' genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.

Adventitious root (AR) formation is indispensable for vegetative asexual propagation. Indole-3-butyric acid (IBA) functioned indirectly as precursor of IAA in regulating AR formation. Ethylene affects auxin synthesis, transport, and/or signaling processes. However, the interactions between auxin and ethylene that control AR formation in apple have not been elucidated. In this study, we investigated the effects of IBA and its interaction with ethylene on AR development in apple. The results revealed that IBA stimulated the formation of root primordia, increased the number of ARs, and upregulated expression of genes (MdWOX11, MdLBD16, and MdLBD29) involved in AR formation. Comparison of different periods of IBA application indicated that IBA was necessary for root primordium formation, while long time IBA treatment obviously inhibited root elongation. RNA-seq analysis revealed that many plant hormone metabolism and signal transduction related genes were differentially expressed. IBA stimulated the production of ethylene during AR formation. Auxin inhibiting ARs elongation depended on ethylene. Together, our results suggest that the inhibitory role of auxin on AR elongation in apples is partially mediated by stimulated ethylene production.

Pre-planting testing of seeds and plantlets for the existence of quarantine pathogens is an important phytosanitary measure. The CRISPR-mediated molecular diagnostic methodologies are being developed for pathogens detection, but many challenges remain. Here, we profiled an engineered Crispr/LbCas12a variant (LbCas12a-5M) that has more robust trans-cleavage activity and a wider PAM sequences (TNTN) preference than wild type. We developed a procedure for screening specific sequences of bacterial plant pathogens, and the designed species-specific crRNA displayed no cross-reactions with other bacterial species. Combined with a simple extraction of bacterial DNA, an LbCas12a-5M-based visual detection technique was established and optimized for detecting quarantine pathogens Erwinia amylovora and Acidovorax citrulli with detection limits up to 40 CFU/reaction and a sensitivity consistent with qPCR assay. This protocol was faster and simpler than qPCR, requiring 40 min or less from sample preparation. We further validated the potential application of the method by showing that it can be used for rapid and accurate diagnosis of A. citrulli on seeds of watermelon, with 100% agreement with the results of qPCR assay. The developed method simplifies the detection of pathogens and provides cost-effective countermeasures to quarantine interventions.

Apple (Malus domestica Borkh.), an economically important fruit crop, is widely cultivated worldwide. However, apple trees often suffer from environmental stressors which affect the quality and yield of apple fruit. Acetylserotonin methyltransferase (ASMT) is a key enzyme in melatonin biosynthesis and is known to play critical roles in melatonin production and stress responses of plants. In this study, a total of 37 ASMT genes in apple were identified and unequally distributed on 10 apple chromosomes, with fragment duplication detected in four pairs of genes. The ASMT genes were classified into three groups based on their sequence compositions and phylogenetic relationship. Sequence analysis showed that apple ASMT genes were generally conserved in each group, with minor variations in gene structure and motif distribution. Transcriptome analysis revealed that most apple ASMT genes were nearly unchanged or downregulated under heat, cold, drought, and NaCl stress, whereas MdASMT6, MdASMT11, MdASMT14 and MdASMT19 were highly induced by these stressors. Subsequent qRT-PCR of four apple ASMT genes showed that MdASMT11 and MdASMT14 were extremely up-regulated under heat, cold, drought, and NaCl stress. They were expressed in all tissues, especially in the roots. Subcellular localization showed that MdASMT11 mainly distributed on cell membranes and MdASMT14 mainly anchored to the nucleus and cell membrane, indicating that MdASMT11 and MdASMT14 are potential genes regulating abiotic stress resistance in apple. These results facilitate further investigation to the functional characterization of MdASMT genes in the synthesis of melatonin and the response to abiotic stress and in apple.

Columnar apples trees, originated from a bud mutation ‘Wijcik McIntosh,’ develop a simple canopy and set fruit on spurs. These characteristics make them an important genetic resource for improvement of tree architecture. Genetic studies have uncovered that columnar growth habit is a dominant trait and is caused by a retroposon insertion that induces the expression of the neighboring gene Co encoding a 2OG-Fe(II) oxygenase. Here we report the genetic mapping of two loci of recessive suppressors (genes) c2 (on Chr10) and c3 (on Chr9) that are linked to repression of the retroposon-induced Co gene expression and associated columnar phenotype in 275 F1 seedlings, which were developed from a reciprocal cross between two columnar selections heterozygous at the Co locus. The mapping was accomplished by sequencing a genomic pool comprising 18 columnar seedlings and another pool of 16 standard seedlings that also carry the retroposon insertion, and by exploring DNA variants of segregation types that are informative for mapping recessive traits in apple. The informative segregation types include , , , , and , where each letter denotes one of the four DNA bases and the letters in bold represent variants in relation to the reference genome. The alleles in each first and third positions are assumed in linkage with the recessive suppressors’ allele in the two parents, respectively. Using RNA-seq analysis, we further revealed that the Co gene together with the differentially expressed genes under loci c2 and c3 formed a co-expression gene-network module associated with growth habit, in which 12 MapMan Bins were enriched.

Short interspersed nuclear elements (SINEs) are non-autonomous retrotransposons that are highly abundant, but not well annotated, in plant genomes. In this study, we identified 41,573 copies of SINEs in seven citrus genomes, including 11,275 full-length copies. The citrus SINEs were distributed among 12 families, with an average full-length rate of 0.27, and were dispersed throughout the chromosomes, preferentially in AT-rich areas. Approximately 18.4% of citrus SINEs were found in close proximity (≤1 kb upstream) to genes, indicating a significant enrichment of SINEs in promoter regions. Citrus SINEs promote gene and genome evolution by offering exons as well as splice sites and start and stop codons, creating novel genes and forming tandem and dispersed repeat structures. Comparative analysis of unique homologous SINE-containing loci (HSCLs) revealed chromosome rearrangements in sweet orange, pummelo, and mandarin, suggesting that unique HSCLs might be valuable for understanding chromosomal abnormalities. This study of SINEs provides us with new perspectives and new avenues by which to understand the evolution of citrus genes and genomes.

Fruit ripening is a complicated process that is influenced by diverse factors at different regulation levels. The microRNAs (miRNAs), a class of endogenous small noncoding RNAs, are recently demonstrated to regulate several critical biological processes in various aspects of the fruit's life cycle. In this paper, to gain more insights into the regulatory mechanism of miRNAs in apple fruit ripening, a genome-wide identification of the sRNAome was implemented in the postharvest ‘Qinguan’ apple fruit. The ripening process of the postharvest apple fruit was significantly inhibited via the application of 1-MCP (1 µL/L). Totally, 569 miRNAs were successfully identified, including 132 known and 437 novel miRNAs, and 29 of them were significantly differentially expressed. The mdm-miR398a, mdm-miR395i-3p, mdm-miR395b, mdm-miR395d-3p, mdm-miR395h, mdm-miR395g-3p, and the novel-miR156 were identified to be the differentially expressed miRNAs. Moreover, after parsing the targets of these miRNAs, several transcription factors, such as ERF and WRKY, were found to be involved in the apple fruit ripening. The above results provide new information for understanding the sophisticated coordinated regulatory network of apple fruit ripening.

Ascorbic acid (AsA) has an important role in plant growth and development, protecting cells against oxidative challenges when exposed to environmental stresses. We investigated the mechanism by which exogenous AsA enhances hypoxia tolerance and alleviates oxidative damage in sensitive and tolerant apple rootstocks ( Malus sieversii and M. hupehensis ). Although such stress inhibited seedling growth in both species, the extent of this effect differed greatly between the two genotypes. Under stress, dry weights were significantly lower from the hypoxia-sensitive M. sieversii than from the controls, but they increased significantly with the addition of AsA. The contents of hydrogen peroxide (H 2 O 2 ) and superoxide radicals (O 2 − ) were significantly increased in roots of both species exposed to hypoxia. This led to oxidative damage, which was indicated by the accumulation of malondialdehyde (MDA). However, that reaction in M. sieversii was significantly suppressed when AsA was supplemented. Exogenously applied AsA enhanced the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in both species, albeit to a greater extent in M. sieversii than in M. hupehensis under hypoxia stress. Exogenously applied AsA raised its endogenous levels of both Malus species exposed to hypoxia, but to a greater extent in M. sieversii. AsA applications also slightly enhanced root glutathione (GSH) contents in stressed plants, but differences were not statistically significant between the treated and untreated plants. AsA possibly enhance hypoxia tolerance by elevating its endogenous level and improving the capacity of the ascorbate–glutathione cycle, thereby decreasing the occurrence of oxidative damage in the membranes.

Plant hormones play important roles in regulating developmental processes and signaling networks involved in plant responses to biotic and abiotic stresses. We comparatively studied the growth and endogenous hormonal levels in leaves and roots in two Malus species (M. sieversii and M. hupehensis) differing in hypoxia tolerance under normoxic and hypoxia stress. The results showed that hypoxia stress inhibited growth of seedlings of both Malus species, but with significant differences in intensity. Exposure to hypoxia altered the levels of endogenous hormones in leaves and roots in both Malus seedlings. Leaf and root abscisic acid (ABA) contents increased in response to hypoxia stress in both genotypes despite different extents. Compared with M. hupehensis, M. sieversii was more responsive to hypoxia stress, resulting in larger increases in leaf and root ABA contents. The changes in leaf and root ABA contents correlating with the different tolerance levels of the genotypes confirm the involvement of this hormone in plant responses to hypoxia stress. Gibberellins (GAs; GA1 + GA4) continuously increased in leaves and roots during the whole period of stress, whereas indole-3-acetic acid (IAA) showed a sharp increase at the early stage in both Malus seedlings. In addition, zeatin riboside (ZR), dihydrozeatin riboside (DHZR), and isopentenyl adenine (IPA) differed in their pattern of changes in both Malus seedlings under hypoxia stress. Based on variations in endogenous hormonal levels in both Malus species that differ in their ability to tolerate hypoxia, we conclude that not a single hormone but multiple hormones and their interplay are responsible for hypoxia tolerance.

The physiological responses and salt tolerance of apple rootstocks were evaluated under high salinity. Seedlings of 15 kinds of apple rootstocks were treated with 200 mM NaCl for 10 d to assess growth, biomass, and physiological responses. Under NaCl stress, their growth parameters decreased compared with the control, with the greatest changes in leaf number, plant height, and relative water content (RWC). In contrast, electrolyte leakage (EL) and activities of peroxidase (POD) and catalase (CAT) were increased. For most rootstocks, stress treatment decreased malondialdehyde (MDA) and soluble protein contents along with superoxide dismutase (SOD) activity. Their degree of salt tolerance was comprehensively evaluated based on salt injury index (SI), adversity resistance coefficients (ARC), and cluster analysis. Malus prunifolia Borkh cv. dongbeihuanghaitang, M. sieboldii Rehd cv. daguohongsanyehaitang, M. prunifolia Borkh cv. qiuzi, and M. xiaojinensis Cheng et Jia were salt-tolerant rootstocks, whereas M. prunifolia Borkh cv. yingyehaitang, M. micromalus Hemsl, and M. sieboldii Rehd cv. lushihongguo were salt-sensitive rootstocks. These differences in sensitivity were associated with variations in the activities of anti-oxidation enzymes and in the amount of organic osmotica.

Variations in biomass productivity, plant water-use efficiency (WUEp), and carbon isotope composition (δ13C) were investigated among 10 Malus rootstocks. In the semi-controlled environmental of a greenhouse, plants were watered to either 75% or 50% of field capacity. For each treatment, significant differences were found in dry matter accumulation and allocation, δ13C, and WUEp. Relative growth rate (RGR) was correlated with WUEp but not with allocation pattern. Variations in whole-plant transpiration were a result of fluctuations in the rate of transpiration per unit leaf area rather than from differences in leaf area or root weight per plant. Values for transpiration per unit leaf area or root weight were lower when the proportion of either leaf area or root weight per unit plant weight was larger. Rootstock differences in δ13C were related to changes in stomatal conductance rather than in net photosynthesis. Finally, δ13C was significantly correlated with WUEp and rootstock rankings based on both of those parameters were maintained regardless of watering treatment.

Seedlings of two Malus species ( M. hupehensis and M. toringoides ) were hydroponically grown in normoxic and hypoxic nutrient solutions to determine their hypoxia tolerance and adaptation of root anaerobic respiration to hypoxia stress. The growth of both species was inhibited under hypoxia stress. During hypoxia stress, the generation of superoxide anion radical (O 2 − ) and content of hydrogen peroxide (H 2 O 2 ) in roots were significantly increased in both species with similar trends, which led to an increase of malondialdehyde (MDA) content and relative membrane permeability (RMP). The degree of growth inhibition and the levels of O 2 − , H 2 O 2 , MDA and RMP were greater in M. toringoides than in M. hupehensis . Under hypoxia stress, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) activities in roots were increased during the first 12 days of hypoxia stress and then gradually decreased in both species. The contents of acetaldehyde, alcohol and lactate in roots were also increased and showed similar trends as the activities of anaerobic respiration enzymes. The increases in PDC and ADH activities and lactate content in M. hupehensis under hypoxia conditions were greater than those of M. toringoides , but alcohol and acetaldehyde contents showed opposite trends. These data suggest that M. hupehensis is more tolerant of hypoxia and had less damage from oxidative stress than M. toringoides under hypoxia stress. The capability for anaerobic respiration is up-regulated in roots of Malus in response to hypoxia stress to minimize damage, and the higher hypoxia tolerance of M. hupehensis may be partly due to the higher enzyme activity of PDC, ADH and LDH and lower accumulation of acetaldehyde and alcohol.

Responses of growth and antioxidant system to root-zone hypoxia stress were comparatively studied in two Malus species (M. hupenensis and M. toringoides) differing in hypoxia tolerance. 50-day-old seedlings were hydroponically grown for 20 days in normoxic and hypoxic nutrient solutions. Hypoxia stress inhibited the growth of both species. Compared with M. hupenensis, M. toringoides was more responsive to hypoxia stress, resulting in larger decreases in leaf number, root length, plant height, and biomass production. The contents of superoxide radicals $$ \left( {{\text{O}}_2 \bar{ \cdot }} \right) $$ and hydrogen peroxide (H2O2) significantly increased in roots of both species exposed to hypoxia stress, and resulted in lipid peroxidation, which was indicated by accumulated concentration of malonaldehyde (MDA). In addition, a significant increase in $$ {\text{O}}_2 \bar{ \cdot } $$ , H2O2 and MDA contents was found in M. toringoides under hypoxia stress. In responses to hypoxia stress, peroxidse (POD), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities increased during the early part of the hypoxia stress, but decreased in the late period; the activities of SOD, POD and APX were more increased in M. hupenensis than in M. toringoides. Ascorbic acid (AsA) and glutathione (GSH) accumulation was also higher in M. hupenensis than in M. toringoides in the early period under hypoxia stress. These results suggest that the hypoxia-tolerant M. hupenensis has a larger protective capacity against oxidative damage by maintaining higher induced activities of antioxidant system than the hypoxia-sensitive M. toringoides.