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Rosaceae is an economically important plant family that can be affected by a multitude of pathogenic microbes, some of which can cause dramatic losses in production. As a type of pattern-recognition receptor, receptor-like proteins (RLPs) are considered vital regulators of plant immunity. Based on genome-wide identification, bioinformatic analysis, and functional determination, we investigated the evolutionary characteristics of RLPs, and specifically those that regulate Valsa canker, a devastating fungal disease affecting apple and pear production. A total of 3028 RLPs from the genomes of 19 species, including nine Rosaceae, were divided into 24 subfamilies. Five subfamilies and seven co-expression modules were found to be involved in the responses to Valsa canker signals of the resistant pear rootstock Pyrus betulifolia ‘Duli-G03’. Fourteen RLPs were subsequently screened as candidate genes for regulation of resistance. Among these, PbeRP23 (Chr13.g24394) and PbeRP27 (Chr16.g31400) were identified as key resistance genes that rapidly enhance the resistance of ‘Duli-G03’ and strongly initiate immune responses, and hence they have potential for further functional exploration and breeding applications for resistance to Valsa canker. In addition, as a consequence of this work we have established optimal methods for the classification and screening of disease-resistant RLPs.

Iron (Fe) deficiency affects plant growth and development. The proton pump interactor (PPI) in plants responds to multiple abiotic stresses, although it has not been well characterized under Fe deficiency stress. In this study, we systematically identified and analyzed the PPI gene family in apple. Three PPI candidate genes were found, and they contained 318-1349 amino acids and 3-7 introns. Under Fe deficiency stress, we analyzed the expression of all the PPI genes in roots of apple rootstock Malus xiaojinensis. Expression of the gene MD11G1247800, designated PPI1, is obviously induced by Fe deficiency treatment in M. xiaojinensis. We first cloned MxPPI1 from M. xiaojinensis and determined its subcellular localization, which indicated that it is localized in the cell membrane and nucleus in tobacco. We found that the level of expression of the MxPPI1 protein increased significantly under Fe deficiency stress in apple calli. Moreover, overexpressing MxPPI1 in apple calli enhanced the activities of ferric chelate reductase and H

Epidermal waxes are part of the outermost hydrophobic structures of apples and play a significant role in enhancing apple resistance and improving fruit quality. The biosynthetic precursors of epidermal waxes are very long-chain fatty acids (VLCFAs), which are made into different wax components through various wax synthesis pathways. In Arabidopsis thaliana, the AtLACS1 protein can activate the alkane synthesis pathway to produce very long-chain acyl CoAs (VLC-acyl-CoAs), which provide substrates for wax synthesis, from VLCFAs. The apple protein MdLACS1, encoded by the MdLACS1 gene, belongs to the AMP-binding superfamily and has long-chain acyl coenzyme A synthase activity, but its function in apple remains unclear. Here, we identified MdLACS1 in apple (Malus × domestica) and analyzed its function. Our results suggest that MdLACS1 promotes wax synthesis and improves biotic and abiotic stress tolerance, which were directly or indirectly dependent on wax. Our study further refines the molecular mechanism of wax biosynthesis in apples and elucidates the physiological function of wax in resistance to external stresses. These findings provide candidate genes for the synergistic enhancement of apple fruit quality and stress tolerance.

The objective of this study was to evaluate the ascorbic acid content in vegetative and generative organs of crabapple from the collection of the M.M. Gryshko National Botanical Garden of National Academy of Sciences of Ukraine. Considering the abundant flowering, higher fruiting in comparison with Malus domestica Borkh., increased content of nutrients and biologically active substances in flowers and leaves of crabapple, they deserve wide use in production. The genotypes were planted to a permanent place of growth in 2014. Total ascorbic acid content in flowers ranged from 116 (Holliana) to 188 (Era) mg / 100 g, in leaves from 112 (Van Ezeltin) to 183 (Royalty) mg / 100 g. The objects of the study are valuable for use as raw materials of biologically active additives. Using the flowers and leaves of Malus spp., As raw materials for the production of dry powder, will meet the needs of consumers for affordable and nutritious ingredients rich in vitamin C.

TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in plant abiotic stresses. However, little is known about the role of TCP genes in the drought stress tolerance of apple. Here, we found that abscisic acid (ABA) and drought treatment reduced the expression of MdTCP46, and overexpression of MdTCP46 reduced ABA sensitivity and drought stress resistance. MdTCP46 was found to interact with MdABI5 both in vitro and in vivo, and this interaction was essential for drought resistance via the ABA-dependent pathway. Overexpression of MdABI5 enhanced ABA sensitivity and drought stress resistance by directly activating the expression of MdEM6 and MdRD29A. MdTCP46 significantly suppressed the transcriptional activity of MdABI5, thereby negatively regulating MdABI5-mediated ABA signalling and drought response. Overall, our results demonstrate that the MdTCP46-MdABI5-MdEM6/MdRD29A regulatory module plays a key role in the modulation of ABA signalling and the drought stress response. These findings provide new insight into the role of MdTCP46 in ABA signalling and abiotic stress responses.

Societal Impact Statement\ud Many economically, culturally, and historically important apple cultivars are triploids, which have three copies of each chromosome instead of the more typical two copies in diploids. Despite their prevalence and importance, there have been conflicting reports regarding their origin and their ability to beget diploids. New genetic analysis methodologies outlined in this study have clarified the genetic origin of triploid apple cultivars and suggest that triploidy has been a dead end in historic apple pedigrees. The specific results of this study have resolved the pedigrees of many cultivars, including the famous English cultivar Cox's Orange Pippin and the oldest known US cultivar Roxbury Russet.\ud \ud Summary\ud In apple (Malus × domestica), most cultivars are diploid, though a sizeable number are triploids, which tend to be stronger growing, more robust, and bear larger fruit. However, triploidy is also associated with strongly reduced fertility. Some recorded pedigrees for historical apple cultivars include triploids as parents of diploids, despite this reputation of poor fertility. This information, coupled with some initiatives using triploids in breeding efforts, result in confusion about how possible or common it is for triploids to be parents of diploid offspring. To date, no studies have systematically evaluated and identified pedigrees of triploid apple cultivars to resolve these contradictions.\ud Here, we describe a method to make triploid genotype calls using Illumina Infinium single nucleotide polymorphism (SNP) array data through a novel Python script: ploidyClassifier. SNP data for 219 unique triploids was compared alongside 2498 unique diploid apple accessions to conduct pedigree reconstruction.\ud Unreduced gamete-donating parents were identified for over half of the triploid accessions. From those, reduced gamete-donating parents were identified for nearly half. Full or partial pedigrees for many classic triploids were uncovered, including that of the oldest known American cultivar, ‘Roxbury Russet’. All tested pedigrees from literature that listed triploids as parents of diploids were deemed false, including that of the well-known ‘Cox's Orange Pippin’, whose previously unreported second parent was also identified here as ‘Rosemary Russet’.\ud These results together suggest that historic triploids are mostly or solely the product of diploid parentage and that triploidy has been a dead end in historic apple pedigrees.

Adventitious root formation is a bottleneck for the mass propagation of microshoots, and nitrate is an essential nutrient regulating adventitious roots. WOX11 is involved in adventitious rooting. But the crosstalk between nitrate and WOX11 is completely unknown. In this study, MdWOX11 transgenic apple microshoots were grown on different nitrate treatments. Low nitrate promotes adventitious rooting in overexpressed microshoots more than wild type and RNA interference microshoots. In contrast, medium nitrate significantly inhibits it in overexpressed and RNA interference microshoots compared with wild type microshoots. Stem anatomy indicated that medium nitrate delays root primordia formation compared with low nitrate. Methyl jasmonate and zeatin riboside played positive and negative roles in adventitious rooting, respectively. Transcriptomic analysis was conducted to understand the molecular mechanisms behind the phenotypes better. Hormone signalling, sugar metabolism, nitrogen metabolism, cell cycle and root development pathway-related genes were selected for their potential involvement in adventitious rooting. Results suggest that nitrogen signaling and MdWOX11 expression affect cytokinin accumulation and response to cytokinin through regulating the expression of genes related to cytokinin synthesis and transduction pathways, which ultimately affect adventitious rooting. This study provided important insights into the complicated networks involved in adventitious rooting in transgenic microshoots under nitrate treatments.

Drought stress is an important factor limiting apple production. γ-Aminobutyric acid (GABA) exists widely in plants and participates in the response to abiotic stress as a metabolite or signaling molecule. The role of exogenous GABA in apple plants, response to long-term drought stress remains unclear. Our study confirmed that exogenous GABA affects the drought resistance of apple plants under long-term drought stress. We found that 1 mM exogenous GABA improved the resistance of apple seedlings to long-term drought stress. The plants showed better growth, less reactive oxygen radical accumulation, less damage to cell membranes and greater active photosynthetic capacity. Under long-term drought stress, exogenous GABA facilitated GABA shunt, resulting in more accumulation of organic acids, namely citric acid, succinic acid and malic acid, in roots and stems of apple seedlings. In addition, exogenous GABA upregulated the expression of cellulose-related genes and lignin-related genes, and activated secondary cell wall-related transcription factors to synthesize more cellulose and lignin. A multiple factorial analysis confirmed that the GABA shunt and the biosynthesis of cellulose and lignin substantially contributed to the growth of apple seedlings with the application of exogenous GABA under long-term drought stress. Our results suggested that exogenous GABA improved the resistance of apple seedlings to long-term drought stress by enhancing GABA shunt and secondary cell wall biosynthesis.

Apple (Malus × domestica Borkh.) cropping behavior, if not regulated, is often manifested by high yields of small-sized fruit in so called ON-years, which are usually followed by strongly reduced crop loads in OFF-years. Such cropping pattern is defined as biennial bearing and causes significant losses in apple production. The growth of apple fruit overlaps with the formation of flower buds, which remain dormant until the following spring. Earlier works proposed that some fruit-derived mobile compounds, as e.g., phytohormones, could suppress flower bud formation that thereby leads to biennial bearing. We addressed this hypothesis by analyzing 39 phytohormones in apple seeds, fruit flesh and by measuring phytohormone export from the fruits of the biennial bearing cultivar ‘Fuji’ and of the regular bearing cultivar ‘Gala’. Moreover, we analyzed the same compounds in bourse buds from fruiting (ON-trees) and non-fruiting (OFF-trees) spurs of both apple cultivars over the period of flower bud formation. Our results showed that apple fruit exported at least 14 phytohormones including indole-3-acetic acid and gibberellin A3; however, their influence on flower bud formation was inconclusive. A gibberellin-like compound, which was detected exclusively in bourse buds, was significantly more abundant in bourse buds from ON-trees compared with OFF-trees. Cultivar differences were marked by the accumulation of trans-zeatin-O-glucoside in bourse buds of ‘Gala’ ON-trees, whereas the levels of this compound in ‘Gala’ OFF were significantly lower and comparable to those in ‘Fuji’ ON- and OFF-trees. Particular phytohormones including five cytokinin forms as well as abscisic acid and its degradation products had higher levels in bourse buds from OFF-trees compared with ON-trees and were therefore proposed as potential promotors of flower bud initiation. The work discusses regulatory roles of phytohormones in flower bud formation in apple based on the novel and to date most comprehensive phytohormone profiles of apple fruit and buds.

Bud dormancy helps woody perennials survive winter and activate robust plant development in the spring. For apple (Malus × domestica), short-term chilling induces bud dormancy in autumn, then prolonged chilling leads to dormancy release and a shift to a quiescent state in winter, with subsequent warm periods promoting bud break in spring. Epigenetic regulation contributes to seasonal responses such as vernalization. However, how histone modifications integrate seasonal cues and internal signals during bud dormancy in woody perennials remains largely unknown. Here, we show that H3K4me3 plays a key role in establishing permissive chromatin states during bud dormancy and bud break in apple. The global changes in gene expression strongly correlated with changes in H3K4me3, but not H3K27me3. High expression of DORMANCY-ASSOCIATED MADS-box (DAM) genes, key regulators of dormancy, in autumn was associated with high H3K4me3 levels. In addition, known DAM/SHORT VEGETATIVE PHASE (SVP) target genes significantly overlapped with H3K4me3-modified genes as bud dormancy progressed. These data suggest that H3K4me3 contributes to the central dormancy circuit, consisting of DAM/SVP and abscisic acid (ABA), in autumn. In winter, the lower expression and H3K4me3 levels at DAMs and gibberellin metabolism genes control chilling-induced release of dormancy. Warming conditions in spring facilitate the expression of genes related to phytohormones, the cell cycle, and cell wall modification by increasing H3K4me3 toward bud break. Our study also revealed that activation of auxin and repression of ABA sensitivity in spring are conditioned at least partly through temperature-mediated epigenetic regulation in winter., 果樹が季節に応答して休眠から目覚めるしくみを解明 --果樹の休眠と発芽におけるヒストン修飾の寄与を解析--. 京都大学プレスリリース. 2022-07-01.

Miniature inverted-repeat transposable elements (MITEs) are widely distributed in the plant genome and can be methylated. However, whether DNA methylation of MITEs is associated with induced allelic expression and drought tolerance is unclear. Here, we identified the drought-inducible MdRFNR1 (root-type ferredoxin-NADP+ oxidoreductase) gene in apple (Malus domestica). MdRFNR1 plays a positive role in drought tolerance by regulating the redox system, including increasing NADP+ accumulation and catalase and peroxidase activities and decreasing NADPH levels. Sequence analysis identified a MITE insertion (MITE-MdRF1) in the promoter of MdRFNR1-1 but not the MdRFNR1-2 allele. MdRFNR1-1 but not MdRFNR1-2 expression was significantly induced by drought stress, which was positively associated with the MITE-MdRF1 insertion and its DNA methylation. The methylated MITE-MdRF1 is recognized by the transcriptional anti-silencing factors MdSUVH1 and MdSUVH3, which recruit the DNAJ domain-containing proteins MdDNAJ1, MdDNAJ2, and MdDNAJ5, thereby activating MdRFNR1-1 expression under drought stress. Finally, we showed that MdSUVH1 and MdDNAJ1 are positive regulators of drought tolerance. These findings illustrate the molecular roles of methylated MITE-MdRF1 (which is recognized by the MdSUVH–MdDNAJ complex) in induced MdRFNR1-1 expression as well as the drought response of apple and shed light on the molecular mechanisms of natural variation in perennial trees.

The K homology (KH) repeat is an RNA-binding motif that exists in various proteins, some of which participate in plant growth. However, the function of KH domain-containing proteins in plant defence is still unclear. In this study, we found that a KH domain-containing protein in apple (Malus domestica), HEN4-like (MdKRBP4), is involved in the plant immune response. Silencing of MdKRBP4 compromised reactive oxygen species (ROS) production and enhanced the susceptibility of apple to Valsa mali, whereas transient overexpression of MdKRBP4 stimulated ROS accumulation in apple leaves, indicating that MdKRBP4 is a positive immune regulator. Additionally, MdKRBP4 was proven to interact with the VmEP1 effector secreted by V. mali, which led to decreased accumulation of MdKRBP4. Coexpression of MdKRBP4 with VmEP1 inhibited cell death and ROS production induced by MdKRBP4 in Nicotiana benthamiana. These results indicate that MdKRBP4 functions as a novel positive regulatory factor in plant immunity in M. domestica and is a virulence target of the V. mali effector VmEP1.

α-Farnesene is a sesquiterpene present in plants. It was first discovered in apples. It plays an important role in the plant defence response and is considered a key factor in the occurrence of superficial scald. The gene encoding α-farnesene synthase, which is the last key enzyme in the biosynthetic pathway of α-farnesene in apple fruit, has become the primary target enzyme for controlling the genetic manipulation of α-farnesene biosynthesis. In this study, the yeast one-hybrid assay and the dual luciferase assay were used to ascertain the relationship between MdLSD1 and MdAFS. Real-time PCR was used to analyse the molecular mechanism underlying the regulation of MdAFS by MdLSD1. Our results revealed that transcription factor MdLSD1, which is closely related to programmed cell death in apple fruit tissues, binds to MdAFS. Transient transformation of apple skin with vectors overexpressing MdLSD1 showed that the gene negatively regulates MdAFS. Overall, we suggest that MdLSD1 negatively regulates MdAFS. Our results are of great significance for future research on the transcriptional regulation of the α-farnesene synthase gene and provide a new direction for exploring the specific mechanism of programmed cell death involved in superficial-scald incidence.

Background and Aims Understanding the mutual co-ordination of vegetative and reproductive growth is important in both agricultural and ecological settings. A competitive relationship between vegetative growth and fruiting is often highlighted, resulting in an apparent trade-off between structural growth and fruit production. However, our understanding of factors driving this relationship is limited. Methods We used four scions grafted onto a series of size-controlling rootstocks to evaluate the relationships between the annual fruit yield and radial growth of trunks, branches and roots. To assess tree radial growth, we measured ring widths on extracted tree cores, which is an approach not frequently used in a horticultural setting. Key Results We found that the yield and radial growth were negatively related when plotted in absolute terms or as detrended and normalized indices. The relationship was stronger in low vigour trees, but only after the age-related trend was removed. In contrast, when trunk radial growth was expressed as basal area increment, the negative relationship disappeared, suggesting that the relationship between trunk radial growth and fruit yield might not be a true trade-off related to the competition between the two sinks. The effect of low yield was associated with increased secondary growth not only in trunks but also in branches and roots. In trunks, we observed that overcropping was associated with reduced secondary growth in a subsequent year, possibly due to the depletion of reserves. Conclusions Our results show that variation in annual fruit yield due to tree ageing, weather cueing and inherent alternate bearing behaviour is reflected in the magnitude of secondary growth of fruit trees. We found little support for the competition/architecture theory of rootstock-induced growth vigour control. More broadly, our study aimed at bridging the gap between forest ecology and horticulture.

Small ubiquitin-related modifier (SUMO)-mediated post-translational protein modification is widely conserved among eukaryotes. SUMOylation refers to the covalent attachment of SUMO to target proteins that alters their function, location, and protein-protein interactions when plants are under abiotic stress. We identified 37 genes in the apple genome that encoded members of the SUMOylation pathway. In addition, RNA-Seq data shows their expression levels between different tissues. We can find that there are mainly expressed genes between each component to ensure that the entire pathway works in the plant. We found that the expression levels of 12 genes were significantly changed under NaCl and ABA treatment through qRT-PCR. MdSIZ1a strongly expression responded to NaCl and ABA treatment. Subsequently, MdSIZ1a was cloned and transformed into apple callus, further verifying the important role of the SUMOylation pathway under stress conditions. The interaction between MdSIZ1a and MdSCEa was verified by yeast two-hybrid, confirming that MdSIZ1a acts as bridge enzyme on MdSCEa and target substrates. Finally, we predicted and analyzed the functional interaction network of E3 ligase to shed light on protein interactions and gene regulatory networks associated with DNA damage repair under abiotic stress in apples.

Jasmonic acid (JA) induces chlorophyll degradation and leaf senescence. B-box (BBX) proteins play important roles in the modulation of leaf senescence, but the molecular mechanism of BBX protein-mediated leaf senescence remains to be further studied. Here, we identified the BBX protein MdBBX37 as a positive regulator of JA-induced leaf senescence in Malus domestica (apple). Further studies showed that MdBBX37 interacted with the senescence regulatory protein MdbHLH93 to enhance its transcriptional activation on the senescence-associated gene MdSAG18, thereby promoting leaf senescence. Moreover, the JA signaling repressor MdJAZ2 interacted with MdBBX37 and interfered with the interaction between MdBBX37 and MdbHLH93, thereby negatively mediating MdBBX37-promoted leaf senescence. In addition, the E3 ubiquitin ligase MdSINA3 delayed MdBBX37-promoted leaf senescence through targeting MdBBX37 for degradation. The MdJAZ2-MdBBX37-MdbHLH93-MdSAG18 and MdSINA3-MdBBX37 modules realized the precise modulation of JA on leaf senescence. In parallel, our data demonstrate that MdBBX37 was involved in abscisic acid (ABA)- and ethylene-mediated leaf senescence through interacting with the ABA signaling regulatory protein MdABI5 and ethylene signaling regulatory protein MdEIL1, respectively. Taken together, our results not only reveal the role of MdBBX37 as an integration node in JA-, ABA- and ethylene-mediated leaf senescence, but also provide new insights into the post-translational modification of BBX proteins.

Proanthocyanidins (PAs) have antioxidant properties and are beneficial to human health. The fruit of apple (Malus × domestica Borkh.), especially the peel, is rich in various flavonoids, such as PAs, and thus is an important source of dietary antioxidants. Previous research on the regulation of PAs in apple has mainly focussed on the transcription level, whereas studies conducted at the post-transcriptional level are relatively rare. In this study, we investigated the function of mdm-miR858, a miRNA with multiple functions in plant development, in the peel of apple fruit. We showed that mdm-miR858 negatively regulated PA accumulation by targeting MdMYB9/11/12 in the peel. During fruit development, mdm-miR858 expression was negatively correlated with MdMYB9/11/12 expression and PA accumulation. A 5'-RACE experiment, GUS staining assays and transient luminescent assays indicated that mdm-miR858 cleaved and inhibited the expression of MdMYB9/11/12. Overexpression of mdm-miR858 in apple calli, tobacco and Arabidopsis reduced the accumulation of PAs induced by overexpression of MdMYB9/11/12. Furthermore, we found that MdBBX22 bound to the mdm-miR858 promoter and induced its expression. Overexpression of MdBBX22 induced the expression of mdm-miR858 to inhibit the accumulation of PAs in apple calli overexpressing MdMYB9/11/12. Under light stress, MdBBX22 induced mdm-miR858 expression to inhibit PA accumulation and thereby indirectly enhanced anthocyanin synthesis in the peel. The present results revealed that the MdBBX22-miR858-MdMYB9/11/12 module regulates PA accumulation in apple. The findings provide a reference for further studies of the regulatory mechanism of PA accumulation and the relationship between PAs and anthocyanins.

Anthocyanins play important roles in plant secondary metabolism. Although previous studies have identified many transcription factors (TFs) that participate in the synthetic pathway of anthocyanins, the regulation mechanism of the pathway remain poorly understood. In this study, we identified a WRKY Group IIc TF, MdWRKY75, which contained a typical WRKYGQK heptapeptide sequence and a C2H2-zinc finger structure. Subcellular localisation assays found that MdWRKY75 was located in the nucleus. Overexpression of MdWRKY75 promoted the accumulation of anthocyanins in apple (Malus domestica L.) ‘Orin’ calli. MdWRKY75 mainly stimulated the accumulation of anthocyanins by binding to the promoter of MYB transcription factor, MdMYB1. Our research could provide new insights into how WRKY TFs regulate the accumulation of anthocyanins in apples.

Conjugation of the small ubiquitin-like modifier (SUMO) peptide to target proteins is an important post-translational modification. SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1 (MdSIZ1) is an apple (Malus domestica Borkh). SUMO E3 ligase that mediates sumoylation of its targets during plant growth and development under adverse environmental conditions. However, it is unclear how MdSIZ1 senses the various environmental signals and whether sumoylation is regulated at the transcriptional level. In this study, we analyzed the MdSIZ1 promoter and found that it contained an MYB binding site (MBS) motif that was essential for the response of MdSIZ1 to low temperature (LT) and drought. Subsequently, we used yeast one-hybridization screening to demonstrate that a MYB transcription factor, MdMYB2, directly bound to the MBS motif in the MdSIZ1 promoter. Phenotypic characterization of MdMYB2 and MdSIZ1 suggested that the expression of both MdMYB2 and MdSIZ1 substantially improved cold tolerance in plants. MdMYB2 was induced by LT and further activated the expression of MdSIZ1, thereby promoting the sumoylation of MdMYB1, a key regulator of anthocyanin biosynthesis in apple. MdMYB2 promoted anthocyanin accumulation in apple fruits, apple calli, and Arabidopsis (Arabidopsis thaliana) in an MdSIZ1-dependent manner. In addition, the interaction of MdMYB2 and the MdSIZ1 promoter substantially improved plant tolerance to cold stress. Taken together, our findings reveal an important role for transcriptional regulation of sumoylation and provide insights into plant anthocyanin biosynthesis regulation mechanisms and stress response.

The homeodomain-leucine zipper (HD-Zip) family has been shown to perform a multitude of functions during plant development and stress responses; however, the family members and functions have not been identified in apple (Malus × domestica). In this study, 83 HD-Zips (MdHDZs) were identified in the apple genome. They were assembled into four subgroups according to the classification in Arabidopsis, where MdHDZs in the same subgroup had similar gene structures and conserved protein motifs. Putative cis-element analysis of MdHDZs promoter regions uncovered numerous elements related to the response of stress and plant hormones. In addition, twelve transcripts of the MdHDZs showed different expression patterns under salt, drought, low temperature and ABA stresses by quantitative reverse transcription-PCR (qRT-PCR) assay. To further explore the function of MdHDZs in apple, MdHDZ3 was selected to verify its function under salt, low temperature and ABA stresses; and genetic transformation was used to obtain MdHDZ3 transgenic apple calli. The results demonstrated that MdHDZ3 increased sensitivity to salt, low temperature and abscisic acid in apple calli, suggesting that MdHDZ3 plays an important role in response to stresses. Subcellular localization and three-dimensional structural analysis revealed that MdHDZ3 was a nuclear-localized protein. Taken together, these findings provide potential information for further identification of HD-Zip proteins in apple.

MicroRNA (miRNA)-mediated gene silencing is a master gene regulatory pathway in plant–pathogen interactions. The differential accumulation of miRNAs among plant varieties alters the expression of target genes, affecting plant defense responses and causing resistance differences among varieties. Botryosphaeria dothidea is an important phytopathogenic fungus of apple (Malus domestica). Malus hupehensis (Pamp.) Rehder, a wild apple species, is highly resistant, whereas the apple cultivar “Fuji” is highly susceptible. Here, we identified a 22-nt miRNA candidate named miRcand137 that compromises host resistance to B. dothidea infection and whose processing was affected by precursor sequence variation between M. hupehensis and “Fuji.” miRcand137 guides the direct cleavage of and produced target-derived secondary siRNA against Ethylene response factor 14 (ERF14), a transcriptional activator of pathogenesis-related homologs that confers disease resistance to apple. We showed that miRcand137 acts as an inhibitor of apple immunity by compromising ERF14-mediated anti-fungal defense and revealed a negative association between miRcand137 expression and B. dothidea sensitivity in both resistant and susceptible apples. Furthermore, MIRCAND137 was transcriptionally activated by the invading fungi but not by the fungal elicitor, implying B. dothidea induced host miRcand137 as an infection strategy. We propose that the inefficient miRcand137 processing in M. hupehensis decreased pathogen-initiated miRcand137 accumulation, leading to higher resistance against B. dothidea.

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in China and other East Asian countries. The plant receptor-like kinase FERONIA is involved in plant cell growth, development, and immunity. However, little is known about the function of FERONIA in apple defence against V. mali. In this study, we found that MdMRLK2 was highly induced by V. mali in twigs of V. mali-susceptible Malus mellana but not in those of the resistant species Malus yunnaensis. 35S:MdMRLK2 apple plants showed compromised resistance relative to wild-type (WT) plants. Further analyses indicated that 35S:MdMRLK2 apple plants had enhanced abscisic acid (ABA) levels and reduced salicylic acid (SA) levels relative to the WT on V. mali infection. MdMRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU), and chitinase (CHT) during V. mali infection. Moreover, MdMRLK2 interacted with MdHIR1, a hypersensitive-induced response protein, and suppressed the MdHIR1-mediated hypersensitive reaction (HR), probably by impairing MdHIR1 self-interaction. Collectively, these findings demonstrate that overexpression of MdMRLK2 compromises Valsa canker resistance, probably by (a) altering ABA and SA levels, (b) suppressing polyphenol accumulation, (c) inhibiting PAL, GLU, and CHT activities, and (d) blocking MdHIR1-mediated HR by disrupting MdHIR1 self-interaction.

Allele-specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar 'Royal Gala' (RG). A high quality chromosome-level genome was assembled using a homozygous tetra-haploid RG plant, derived from anther cultures. Using RNA-sequencing (RNA-seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of 'Golden Delicious' (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele-specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele-specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome-wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

Nitric oxide (NO) is known to modulate the action of several phytohormones. This includes the gaseous hormone ethylene, but the molecular mechanisms underlying the effect of NO on ethylene biosynthesis are unclear. Here, we observed a decrease in endogenous NO abundance during apple (Malus domestica) fruit development and exogenous treatment of apple fruit with a NO donor suppressed ethylene production, suggesting that NO is a ripening suppressor. Expression of the transcription factor MdERF5 was activated by NO donor treatment. NO induced the nucleocytoplasmic shuttling of MdERF5 by modulating its interaction with the protein phosphatase, MdPP2C57. MdPP2C57-induced dephosphorylation of MdERF5 at Ser260 is sufficient to promote nuclear export of MdERF5. As a consequence of this export, MdERF5 proteins in the cytoplasm interacted with and suppressed the activity of MdACO1, an enzyme that converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The NO-activated MdERF5 was observed to increase in abundance in the nucleus and bind to the promoter of the ACC synthase gene MdACS1 and directly suppress its transcription. Together, these results suggest that NO-activated nucleocytoplasmic MdERF5 suppresses the action of ethylene biosynthetic genes, thereby suppressing ethylene biosynthesis and limiting fruit ripening.

Ethylene (ETH) controls climacteric fruit ripening and can be triggered by osmotic stress. However, the mechanism regulating ETH biosynthesis during fruit ripening and under osmotic stress is largely unknown in apple (Malus domestica). Here, we explored the roles of SnRK2 protein kinases in ETH biosynthesis related to fruit ripening and osmoregulation. We identified the substrates of MdSnRK2-I using phosphorylation analysis techniques. Finally, we identified the MdSnRK2-I-mediated signaling pathway for ETH biosynthesis related to fruit ripening and osmoregulation. The activity of two MdSnRK2-I members, MdSnRK2.4 and MdSnRK2.9, was significantly upregulated during ripening or following mannitol treatment. Overexpression of MdSnRK2-I increased ETH biosynthesis under normal and osmotic conditions in apple fruit. MdSnRK2-I phosphorylated the transcription factors MdHB1 and MdHB2 to enhance their protein stability and transcriptional activity on MdACO1. MdSnRK2-I also interacted with MdACS1 and increased its protein stability through two phosphorylation sites. The increased MdACO1 expression and MdACS1 protein stability resulted in higher ETH production in apple fruit. In addition, heterologous expression of MdSnRK2-I or manipulation of SlSnRK2-I expression in tomato (Solanum lycopersicum) fruit altered fruit ripening and ETH biosynthesis. We established that MdSnRK2-I functions in fruit ripening and osmoregulation, and identified the MdSnRK2-I-mediated signaling pathway controlling ETH biosynthesis.

Anthocyanin is one of the most important pigments and nutrients in fruits. Genes encoding R2R3-MYB transcription factors (TFs) are key to anthocyanin regulation. R2R3-MYB activators have been widely studied in the anthocyanin synthesis pathway. However, the mechanism by which R2R3-MYB repressors negatively regulate anthocyanin synthesis remains poorly understood. In the present study, we characterized a subfamily 1 R2R3-MYB anthocyanin repressor gene, MYB306-like, from apple (Malus × domestica) and demonstrated its significance in anthocyanin regulation. The MdMYB306-like protein activates the expression of an anthocyanin repressor gene, MdMYB17, and inhibits the expression of MdDFR (an anthocyanin structural gene) by directly binding to the corresponding promoters. The MdMYB306-like protein interacts with MdMYB17 and MdbHLH33 through its N-terminus. In addition, MdMYB306-like interacts with MdbHLH33 and MdMYB17 to enhance its regulatory activities on MdMYB17 and MdDFR, respectively. Our results revealed that the MdMYB306-like protein, in conjunction with MdMYB17 and MdbHLH33, forms a key regulatory module to fine-tune anthocyanin synthesis in apple.

Ring rot is a destructive apple disease caused by Botryosphaeria dothidea. The resistance mechanism of apple plants to B. dothidea remains unclear. Here, we show that APPLE VACUOLAR PROCESSING ENZYME 4 (MdVPE4) is involved in resistance to B. dothidea. MdVPE4 silencing reduced fruit disease resistance, whereas its overexpression improved resistance. Gene expression analysis revealed that MdVPE4 influenced the expression of fruit disease resistance-related genes, such as APPLE POLYGALACTURONASE 1 (MdPG1), APPLE POLYGALACTURONASE INHIBITOR PROTEIN 1 (MdPGIP1), APPLE ENDOCHITINASE 1 (MdCHI1), and APPLE THAUMATIN-LIKE PROTEIN 1 (MdTHA1). The expression of the four genes responding to B. dothidea infection decreased in MdVPE4-silenced fruits. Further analysis demonstrated that B. dothidea infection induced MdVPE4 expression and enzyme activation in apple fruits. Moreover, MdVPE4 activity was modulated by apple cysteine proteinase inhibitor 1 (MdCPI1), which also contributed to resistance towards B. dothidea, as revealed by gene overexpression and silencing analysis. MdCPI1 interacted with MdVPE4 and inhibited its activity. However, MdCPI1 expression was decreased by B. dothidea infection. Taken together, our findings indicate that the interaction between MdVPE4 and MdCPI1 plays an important role in modulating fruit disease resistance to B. dothidea.

The enterobacterial pathogen Erwinia amylovora uses multiple virulence-associated traits to cause fire blight, a devastating disease of apple and pear trees. Many virulence-associated phenotypes have been studied that are critical for virulence and pathogenicity. Despite the in vitro testing that has revealed how these systems are transcriptionally regulated, information on when and where in infected tissues these genes are being expressed is lacking. Here, we used a high-throughput sequencing approach to characterize the transcriptome of E. amylovora during disease progression on apple flowers under field infection conditions. We report that type III secretion system genes and flagellar genes are strongly co-expressed. Likewise, genes involved in biosynthesis of the exopolysaccharide amylovoran and sorbitol utilization had similar expression patterns. We further identified a group of 16 genes whose expression is increased and maintained at high levels throughout disease progression across time and tissues. We chose five of these genes for mutational analysis and observed that deletion mutants lacking these genes all display reduced symptom development on apple shoots. Furthermore, these induced genes were over-represented for genes involved in sulphur metabolism and cycling, suggesting the possibility of an important role for maintenance of oxidative homeostasis during apple flower infection.

Apple (Malus × domestica) is one of the most important fruit crops grown commercially in temperate regions. The present study was conducted to gain knowledge on genetic variability pertaining to flowering, maturity and some morphological characters. Earliest initial and full bloom was observed in Benoni whereas ASP-69 was the last to bloom. Irish peach was found to mature earliest (88 DAFB) while Maharaji (176 DAFB) was the last to mature. The maximum average leaf blade length (101.00 mm) was observed in ASP-4 and ASP-10. Lal Ambri recorded maximum leaf blade width (67.66 mm). Minimum leaf blade length (58.66 mm) and width (36.0 mm) were recorded in Scarlet Siberean. Average length of fruits varied between 28.1 mm (Scarlet Siberean) and 85.2 mm (Lal Ambri). The maximum average fruit weight ranged from 14.06 g (Scarlet Siberean) to 202.73 g (Lal Ambri). Fruit firmness was maximum in Scarlet Siberean (9.43 kg/cm2) followed by 9.06 kg/cm2 in Ambri, minimum fruit firmness was recorded in June eating (5.35 kg/cm2). TSS ranged from 10.76 ⁰Brix (June Eating) to 14.93⁰Brix (Shalimar Apple-1). The maximum acidity was recorded in Braeburn (0.33%) while minimum (0.12%) in Ambri and ASP-69. As per Mahalanobis D2 analysis, the cultivars were grouped into 3 clusters. Cluster I (36 cultivars) comprised of the highest number of cultivars followed by cluster II (3 cultivars) and cluster III was monogenic with 1 cultivar. Wide variation existed in the germplasm under study which could be made use of in future breeding populations

Although open-central canopy (OCC) is popular in apple (Malus × domestica) orchards in Loess Plateau of China, its relevant photosynthetic mechanisms have not been elucidated. In this study, changes in photosynthetically active radiation (PAR), gas exchange, chlorophyll fluorescence quenching and fruit quality in apple trees were measured in OCC and compared with those in the conventional condensed round and large canopy (RLC). Results showed that light intercepted at different orientations was 44% higher by OCC than that by RLC. The improved light environment within OCC remarkably increased leaf maximum net photosynthetic rate (Pnmax) and significantly decreased stomatal limitation. Under high light, the ratio of photorespiratory rate to gross photosynthetic rate (Pr/Pg) in OCC was higher than that in RLC. Moreover, reversible component in non-photochemical quenching (r(qE)) was increased, while irreversible component (r(qI)) was decreased in OCC than in RLC. As a result, the fruit quality in OCC was greatly boosted as evidenced by the significantly increased single fruit weight, fruit flesh firmness and fruit soluble solid contents and the sharply decreased fruit titratable acid contents. PAR intercepted by the canopy and the fruit soluble solid contents, leaf Pnmax or single fruit weight were positively correlated, while PAR or Pnmax was negatively correlated with the fruit titratable acid contents. Accordingly, the improved crown light environment and the enhanced leaf photosynthetic performance and photoprotective capacity in OCC led to the boosted fruit quality.

Although a few apple (Malus × ×domestica) varieties are self-compatible, little is known about the differences in fruit quality between self- and cross-pollinated apple. In our current study, we compared the fruit quality of self-pollinated apple plants (cultivar 'Hanfu') in self-pollination or cross-pollinated by another cultivar 'Qinguan'. Analysis of fruit quality revealed substantial differences in the external qualities between self- and cross-pollinated apple fruit, but not in the internal qualities. Fruits harvested from self-pollinated 'Hanfu' were smaller and more asymmetrical than those harvested from the cross-pollinated plants. We developed a mathematical model describing how seed number and distribution affect fruit growth. According to this model, the fewer the seeds, the greater the force released from the seeds and the more asymmetrical the fruit. Detection of endogenous hormone and the associated gene expression revealed that gibberellin (GA) levels and GA transporter gene expression on the seedless side were significantly lower than those on the seeded side. Analysis of fruit pectin methylesterase activity and demethylated pectin levels indicated that the lack of GA limits fruit cell wall extension. Additionally, spraying the self-pollinating plants with gibberellic acid increased the fruit weight and lowered the proportion of asymmetrical fruit, recovering the exterior fruit quality to that of the cross-pollinated fruit. Furthermore, exogenous GA treatment increased the wax layer thickness and reduced the fruit water loss rate, leading to a dramatic improvement in fruit storage capacity. Therefore, exogenous GA treatment could be used to ensure regular fruit production of self-pollinated 'Hanfu'.

Dopamine is a catecholamine and an anti-oxidant which functions in responses to stress and it interacts with plant hormones to mediate plant development. At present, there are few studies on the functions of dopamine in apple. This study developed a method for dopamine determination which was used to analyze dopamine in Malus germplasm, in order to clarify the tissue distribution, developmental changes, diurnal variations, and stress responses in apple trees. First, the proposed method was verified. The linear range of quantification was robust from 0.1 to 20 ng mL−1. The instrumental, inter-day precision, and sample repeatability relative standard deviations were 1.024, 5.607, and 7.237%, respectively. The spiked recovery was greater than 100%, indicating the feasibility of the method and its suitability for the rapid analysis of dopamine in Malus. Next, the dopamine content was measured in 322 Malus tissues. The results showed that the dopamine level in Malus was low and the average dopamine content in leaf was higher than in peel and flesh. The dopamine had a skewed distribution that deviated to the right in cultivars and wild accessions. Finally, the tissue specificity, developmental changes, diurnal changes, and responses to stress were analyzed. In cultivar ‘Pinova’ (Malus domestica), the dopamine concentration was the highest in leaf buds and lowest in flesh. The dopamine contents in leaf and flesh decreased with the growth and development of cultivar ‘Liangxiang’ (Malus domestica). The dopamine content of apple leaves was higher after either drought or salinity stress as compared to the control. In this study, a dopamine detection method for apple was established based on HPLC-MS and shown to be a robust approach. This study provides a framework for future research on elucidating the tissue distribution, developmental changes, diurnal variation, and stress responses of dopamine in apple trees.

Out of five wild Malus species in the world, Malus baccata (L.) Borkh (MB) is reported from the extremely cold areas of north western Himalayas. These are widely used as a rootstock or source of resistance against various biotic and abiotic stresses due to its ecological adaptability and cold hardiness. The study of genetic diversity is an important aspect to be covered for proper utilization of the wild apple germplasm for breeding purpose. The 29 Malus baccata accessions were used to divulge their genetic diversity using morpho-molecular markers. Based on morphological characters, Mahalanobis D2statistics resolved all accessions in four clusters. A set of 85 SSR were used to study molecular diversity among the accessions. The AMOVA stated genetic diversity between populations of genotypes was lower (7%) compared to that observed within and among the individuals of the populations (93%). The effective allele number (2.63) was found to be lower than mean allele number (3.7), with mean heterozygosity as 0.51. The gene diversity varied between 0.10 for Hi02d05 to 0.79 for CH-Vf1.The expected heterozygosity was greater than observed heterozygosity for total analysed loci of SSR. The average PIC value was found as 0.50. The accessions were segregated into two distinct classes using heirarchical clustering method. Further, population structure also differentiated the 29 accessions into two populations, with sub-population I accounts for 18 accessions and sub-population II consisted of only 9 accessions. MB-5 and MB-20 accessions fall in the admixture group. The differentiated populations were validated and reconfirmed by the PCoA analysis. . The morphological features and molecular markers revealed significant level of genetic diversity along with population differentiation among the maintained accessions.

To investigate the roles of Harvista (a sprayable 1-methylcyclopropene, 1-MCP, available component is 150 g/ha) and Ethephon (1 mL/L) applied at preharvest in apple (Malus domestica Borkh. cv. Golden Delicious) fruit cuticular wax biosynthesis, the expression of genes related to fruit cuticular wax biosynthesis and ethylene biosynthesis and signaling, ethylene production rate, respiration rate, wax constituents and structure, and fruit quality were determined at harvest and during cold storage. The results showed that 1-MCP inhibited the expression levels of ethylene biosynthesis and signaling-related genes, decreased fruit ethylene production, and inhibited the expression of MdLACS1, MdCER6, MdCER4 and MdWSD1, which resulted in decreases in alcohols, acids, and esters content in fruit cuticular wax. 1-MCP also reduced fruit dropping rate from 17.17% to 12%; maintained fruit firmness, soluble solids, titratable acidity during cold storage; showed about as one fifth in the total length and one third in the widest width of wax crack as that in control at harvest. In contrast, Ethephon produced the opposite effects. In conclusion, 1-MCP inhibited fruit ethylene biosynthesis and signaling, and influenced fruit cuticular wax biosynthesis. Thus, the fruit cuticular wax constituents and structure was altered, and the fruit quality were maintained at harvest and during cold storage. The results provide a new technology for improving apple fruit harvest and postharvest quality by preharvest application of sprayable 1-MCP.

Ubiquitination is an important post-translational protein modification. Although BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC and TRANSCRIPTION ADAPTOR PUTATIVE ZINC FINGER domain protein 2 (BT2) is involved in many biological processes, its role in apple (Malus domestic) root formation remains unclear. Here, we revealed that MdBT2 inhibits adventitious root (AR) formation through interacting with AUXIN RESPONSE FACTOR8 (MdARF8) and INDOLE-3-ACETIC ACID INDUCIBLE3 (MdIAA3). MdBT2 facilitated MdARF8 ubiquitination and degradation through the 26S proteasome pathway and negatively regulated GRETCHEN HAGEN 3.1 (MdGH3.1) and MdGH3.6 expression. MdARF8 regulates AR formation through inducing transcription of MdGH3s (MdGH3.1, MdGH3.2, MdGH3.5, and MdGH3.6). In addition, MdBT2 facilitated MdIAA3 stability and slightly promoted its interaction with MdARF8. MdIAA3 inhibited AR formation by forming heterodimers with MdARF8 as well as other MdARFs (MdARF5, MdARF6, MdARF7, and MdARF19). Our findings reveal that MdBT2 acts as a negative regulator of AR formation in apple.

As apple fruits (Malus domestica) mature, they accumulate anthocyanins concomitantly with losing chlorophyll (Chl); however, the molecular pathways and events that coordinate Chl degradation and fruit coloration have not been elucidated. We showed previously that the transcription factor ETHYLENE RESPONSE FACTOR17 (MdERF17) modulates Chl degradation in apple fruit peels and that variation in the pattern of MdERF17 serine (Ser) residues is responsible for differences in its transcriptional regulatory activity. Here, we report that MdERF17 interacts with and is phosphorylated by MAP KINASE4 (MdMPK4-14G). Phosphorylation of MdERF17 at residue Thr67 by MdMPK4-14G is necessary for its transcriptional regulatory activity and its regulation of Chl degradation. We also show that MdERF17 mutants with different numbers of Ser repeat insertions exhibit altered phosphorylation profiles, with more repeats increasing its interaction with MdMPK4. MdMPK4-14G can be activated by exposure to darkness and is involved in the dark-induced degreening of fruit peels. We also demonstrate that greater phosphorylation of MdERF17 by MdMPK4-14G is responsible for the regulation of Chl degradation during light/dark transitions. Overall, our findings reveal the mechanism by which MdMPK4 controls fruit peel coloration.

Anthocyanin production in apple (Malus domestica) fruit and their consequent coloration can be induced by high-light treatment. The hormone ethylene is also essential for this coloration, but the regulatory relationships that link ethylene and light with anthocyanin-associated coloration are not well defined. In this study, we observed that high-light treatment of apple fruit increased anthocyanin accumulation more than moderate-light treatment did and was the main contributor of induced ethylene production and activation of anthocyanin biosynthesis. A transcriptome study of light-treated apple fruit suggested that a long noncoding RNA (lncRNA), MdLNC610, the corresponding gene of which is physically located downstream from the 1-aminocyclopropane-1-carboxylate oxygenase (ACO) ethylene biosynthesis gene MdACO1, likely affects anthocyanin biosynthesis under high-light treatment. Expression and promoter β-glucuronidase reporter analyses further showed that MdLNC610 upregulates expression of MdACO1 and so likely participates in high-light-induced ethylene biosynthesis. Overexpression of MdACO1 and MdLNC610 in apple fruit and calli indicated that a major increase in MdLNC610 expression activates MdACO1 expression, thereby causing an increase in ethylene production and anthocyanin levels. These results suggest that MdLNC610 participates in the regulation of high-light-induced anthocyanin production by functioning as a positive regulator to promote MdACO1 expression and ethylene biosynthesis. Our study provides insights into the relationship between mRNA and lncRNA networks in the ethylene biosynthetic pathway and anthocyanin accumulation in apple fruit.

International audience; Tree architecture shows large genotypic variability, but how this affects water-deficit responses is poorly understood. To assess the possibility of reaching ideotypes with adequate combinations of architectural and functional traits in the face of climate change, we combined high-throughput field phenotyping and genome-wide association studies (GWAS) on an apple tree (Malus domestica) core-collection. We used terrestrial light detection and ranging (T-LiDAR) scanning and airborne multispectral and thermal imagery to monitor tree architecture, canopy shape, light interception, vegetation indices and transpiration on 241 apple cultivars submitted to progressive field soil drying. GWAS was performed with single nucleotide polymorphism (SNP)-by-SNP and multi-SNP methods. Large phenotypic and genetic variability was observed for all traits examined within the collection, especially canopy surface temperature in both well-watered and water deficit conditions, suggesting control of water loss was largely genotype-dependent. Robust genomic associations revealed independent genetic control for the architectural and functional traits. Screening associated genomic regions revealed candidate genes involved in relevant pathways for each trait. We show that multiple allelic combinations exist for all studied traits within this collection. This opens promising avenues to jointly optimize tree architecture, light interception and water use in breeding strategies. Genotypes carrying favourable alleles depending on environmental scenarios and production objectives could thus be targeted.

In this study, we aimed to elucidate the effect of pulp cell wall structure on fruit hardness and crispness in apples. To this end, we studied the cell wall polysaccharides in two apple varieties, "Hanfu" and "Honeycrisp," during fruit development. Compared with Hanfu, the crispness of Honeycrisp was higher, whereas its harness was lower. The intensity and distribution of immunofluorescence signals indicated that galactose and arabinose contributed to the higher hardness of Hanfu, whereas arabinose, egg-box structure, and fucosylated xyloglucans, distributed in the corners of tricellular junctions, enhanced the cell-cell adhesion and improved the crispness of Honeycrisp. Besides, fucosylated xyloglucan played an important role in promoting the formation and maintaining the strength of the cell wall skeleton and, consequently, retaining the fruit crispness. The esterification state of pectin had little effect on the fruit hardness and crispness in both varieties. Collectively, our findings provided information on the underlying mechanism of fruit texture formation in apples.

In plants, ammonium (NH

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

Iron (Fe) is an essential element for plant growth and development. Fe deficiency can trigger leaf chlorosis and reduce fruit yield. Therefore, it is necessary to explore transcription factors in response to Fe deficiency stress. A total of 29 MhR2R3-MYB transcription factors were identified based on the transcriptome of Malus halliana under Fe deficiency stress. A comprehensive analysis of physical and chemical properties, gene structures, conserved motif composition, evolutionary relationship and chromosome distribution was performed. Subsequently, based on the transcriptome, 14 genes with the most significant expression under Fe deficiency stress were screened for qRT-PCR verification. Among them,the functional characteristics of MhR2R3-MYB4 (MD05G1089600) were further studied in Arabidopsis thaliana. Expression of 13 out of these 14 genes was upregulated, only one was downregulated. Maximum upregulation of MhR2R3-MYB4 under Fe deficiency was 36.39-fold and 58.21-fold compared with day 0 in leaves and roots, respectively. Overexpression of MhR2R3-MYB4 enhanced tolerance to Fe deficiency in A. thaliana and led to multiple biochemical changes: transgenic lines have higher chl a, chl b and Fe

Drought stress tolerance is a complex trait regulated by multiple factors. Here, we demonstrate that the miRNA160–Auxin Response Factor 17 (ARF17)–HYPONASTIC LEAVES 1 module is crucial for apple (Malus domestica) drought tolerance. Using stable transgenic plants, we found that drought tolerance was improved by higher levels of Mdm-miR160 or MdHYL1 and by decreased levels of MdARF17, whereas reductions in MdHYL1 or increases in MdARF17 led to greater drought sensitivity. Further study revealed that modulation of drought tolerance was achieved through regulation of drought-responsive miRNA levels by MdARF17 and MdHYL1; MdARF17 interacted with MdHYL1 and bound to the promoter of MdHYL1. Genetic analysis further suggested that MdHYL1 is a direct downstream target of MdARF17. Importantly, MdARF17 and MdHYL1 regulated the abundance of Mdm-miR160. In addition, the Mdm-miR160-MdARF17–MdHYL1 module regulated adventitious root development. We also found that Mdm-miR160 can move from the scion to the rootstock in apple and tomato (Solanum lycopersicum), thereby improving root development and drought tolerance of the rootstock. Our study revealed the mechanisms by which the positive feedback loop of Mdm-miR160-MdARF17-MdHYL1 influences apple drought tolerance.

In this study, 'Golden Delicious' apples were dipped with γ-aminobutyric acid (GABA) solution to investigate the changes of quality parameters, ethylene anabolism, polyamine metabolism and GABA shunt. Results showed that GABA distinctly suppressed respiratory rate, reduced titratable acidity, maintained higher soluble solid content and pericarp firmness of apples. Compared to the control, GABA also repressed the activities and gene expressions of polyamine oxidase (PAO) and diamine oxidase (DAO), enhanced MdMT, MdMS, MdSAMS, MdSAMDC, MdSPDS, MdODC, MdADC, and MdACL5 expressions, and accelerated the accumulation of putrescine, spermidine, and spermine in the exocarp of apples. Moreover, GABA decreased ethylene release, MdACS and MdACO gene expressions in the exocarp. In addition, exogenous GABA activated MdGAD, MdGDH, MdGS expressions and inhibited MdGABA-T and MdSSADH expressions in the GABA shunt, therefore increased endogenous GABA, pyruvic acid and glutamate contents in the exocarp. These findings suggest that exogenous GABA regulates ethylene anabolism, polyamine metabolism and GABA shunt to maintain fruit quality of 'Golden Delicious' apples.

Twenty-eight apple cultivars were selected for their potential for hard cider production in Ontario; their juice characteristics were measured in 2017 and 2018, beginning two years after planting in 2015. After being harvested and pressed, each juice sample underwent analyses to determine soluble solids concentration (SSC), titratable acidity (TA), pH, yeast assimilable nitrogen (YAN), and polyphenolic concentration. Soluble solids concentration ranged from 10.6 °Brix in Brown’s Apple to 18.3 °Brix in Ashmead’s Kernel. Titratable acidity ranged from 31 as mg malic acid per 100 mL juice in Sweet Alford to 191 as mg malic acid per 100 mL juice in Bramley’s Seedling. The pH ranged from 2.88 in Breakwell to 4.76 in Sweet Alford. Yeast assimilable nitrogen concentration ranged from 60 mg YAN·L−1juice in Medaille d’Or to 256 mg YAN·L−1juice in Bulmer’s Norman. Polyphenols in juice ranged from 131 μg gallic acid equivalents (gae)·mL−1juice in Tolman Sweet to 1042 μg gae·mL−1juice in Stoke Red. Firmness ranged from 6.3 kg in Yarlington Mill to 11.7 kg in GoldRush. The relationships between these variables were also analyzed, showing a connection between acidity and juicing efficiency as well as a relationship between polyphenol concentration and fruit weight. Exploratory analyses indicated that juice attributes can be used to distinguish between cultivars and their origins. Cider producers can use these data to determine what to expect in juice from these cultivars.

China is a center of diversity for Malus Mill. with 27 native species including 21 wild species and six domesticated species. We applied a set of 19 simple sequence repeat markers to genotype 798 accessions of 17 species (12 wild species and five cultivated species) of Malus originating from 14 provinces in China. A total of 500 alleles were detected. Diversity statistics indicated a high level of genetic variation as quantified by the average values of the effective allele number (Ne), expected heterozygosity (He), and Shannon’s Information Index (I) (10.309, 0.886, and 2.545, respectively). Malus sieversii (MSR; He=0.814, I=2.041, Ne=6.054), M. baccata (MBB; He=0.848, I=2.350, Ne=8.652), M. toringoides (MTH; He=0.663, I=1.355, Ne=3.332), and M. hupehensis (MHR; He=0.539, I=0.912, Ne=0.579) showed a higher level of genetic diversity in this study than the previous studies. MSR and MBB contributed to the origin and evolution of some accessions of M. domestica subsp. chinensis (MDC). However, other accessions of MDC showed a closer genetic distance with MBB and cultivated species, especially M. robusta (MRB), M. asiatica (MAN), and M. prunifolia (MPB). Not all accessions of MDC were descended from MSR in Xinjiang Uygur Autonomous Region of China. This research provides novel insights into the genetic relationships of Malus native to China, which will be useful for genetic association studies, germplasm conservation, and breeding programs.

Twenty-eight apple cultivars were selected for their potential for hard cider production in Ontario. An experiment was conducted to evaluate their horticultural potential in the province. After being planted in spring 2015, the trees were evaluated annually for their survival, tree height and spread, trunk growth, flowering dates, flower counts, fruit per tree, pre-harvest drop, crop load, fruit weight, fruit firmness, juicing extraction efficiency, and harvest dates. These horticultural attributes were sufficient to discriminate between cultivars. Additional exploratory analyses indicated a relationship between horticultural attributes and a cultivar’s origin, with British cider cultivars blooming the latest, American cider apples producing the most juice, and French cider cultivars having the highest pre-harvest fruit drop. Cultivars in this study that show promise for continued research in Ontario include Binet Rouge, Bramley’s Seedling, Breakwell, Bulmer’s Norman, Calville Blanc d’Hiver, Cline Russet, Cox Orange Pippin, Crimson Crisp®, Dabinett, Enterprise, Esopus Spitzenberg, Golden Russet, GoldRush, Medaille d’Or, Porter’s Perfection, and Stoke Red.

BackgroundScab, caused by the biotrophic fungusVenturia inaequalis, is the most economically important disease of apples worldwide. During infection,V. inaequalisoccupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth byV. inaequalis, as well as in activating, suppressing, or circumventing resistance protein-mediated defences in apple, is required.ResultsWe generated the first comprehensive RNA-seq transcriptome ofV. inaequalisduring colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid, or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector fromLeptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi.ConclusionsOur study reveals the transcriptomic profile underpinning subcuticular growth byV. inaequalisand provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.

Background GRAS genes formed one of the important transcription factor gene families in plants, had been identified in several plant species. The family genes were involved in plant growth, development, and stress resistance. However, the comparative analysis of GRAS genes in Rosaceae species was insufficient. Results In this study, a total of 333 GRAS genes were identified in six Rosaceae species, including 51 in strawberry (Fragaria vesca), 78 in apple (Malus domestica), 41 in black raspberry (Rubus occidentalis), 59 in European pear (Pyrus communis), 56 in Chinese rose (Rosa chinensis), and 48 in peach (Prunus persica). Motif analysis showed the VHIID domain, SAW motif, LR I region, and PFYRE motif were considerably conserved in the six Rosaceae species. All GRAS genes were divided into 10 subgroups according to phylogenetic analysis. A total of 15 species-specific duplicated clades and 3 lineage-specific duplicated clades were identified in six Rosaceae species. Chromosomal localization presented the uneven distribution of GRAS genes in six Rosaceae species. Duplication events contributed to the expression of the GRAS genes, and Ka/Ks analysis suggested the purification selection as a major force during the evolution process in six Rosaceae species. Cis-acting elements and GO analysis revealed that most of the GRAS genes were associated with various environmental stress in six Rosaceae species. Coexpression network analysis showed the mutual regulatory relationship between GRAS and bZIP genes, suggesting the ability of the GRAS gene to regulate abiotic stress in woodland strawberry. The expression pattern elucidated the transcriptional levels of FvGRAS genes in various tissues and the drought and salt stress in woodland strawberry, which were verified by RT-qPCR analysis. Conclusions The evolution and functional analysis of GRAS genes provided insights into the further understanding of GRAS genes on the abiotic stress of Rosaceae species.

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CCR -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.