MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes.

Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach to their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, the electrophoretic mobility shift assay, and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.

Genome-Wide Identification, Classification, and Expression Analysis of the HD-Zip Transcription Factor Family in Apple (Malus domestica Borkh.).

Homeodomain-leucine zipper (HD-Zip) family genes are considered to play an important role in plant growth and stress tolerance. However, a genome-wide analysis of HD-Zip genes in apples (Malus domestica Borkh.) has not been performed. We detected 48 MdHDZ genes in the apple genome, and categorized them into three subfamilies on the basis of phylogenetic analysis. The chromosomal locations, gene/protein structures, and physiological and biochemical properties of these genes were analyzed. Synteny analysis revealed that segmental duplications were key in the expansion of the apple HD-Zip family. According to an analysis of cis-regulatory elements and tissue-specific expression patterns, MdHDZ genes may be widely involved in the regulation of apple growth and tolerance to environmental stresses. Furthermore, the transcript levels of apple HD-Zip I and II genes were up-regulated in response to fungal treatments. Expression of apple HD-Zip Ⅲ genes was enhanced during adventitious bud regeneration. This suggested possible roles of these genes in regulating the apple response to fungal infection, as well as adventitious bud regeneration. The current results may help us to better understand the evolution and function of apple HD-ZIP genes, and thus facilitate further research on plant resistance to fungal infection and in vitro regeneration.

Transposon insertions regulate genome-wide allele-specific expression and underpin flower colour variations in apple (Malus spp.).

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.

Genome-wide identification and characterization of AINTEGUMENTA-LIKE (AIL) family genes in apple (Malus domestica Borkh.).

AINTEGUMENTA-LIKE (AIL) genes play a key role in various growth and developmental processes in plants. Thus far, the genome-wide identification of AIL genes has been reported for some plant species. However, genome-wide identification of AIL genes has not been conducted in apple (Malus domestica Borkh.). The current study focused on a comprehensive analysis of the AIL genes in the apple genome (i.e., MdAIL genes). In total, 27 MdAIL genes in the apple genome were identified and then divided into four groups according to phylogenetic analysis. The chromosomal locations, gene and protein structures, and physicochemical characteristics of MdAIL genes were analyzed. Synteny analysis revealed that segmental duplication events played a major role in the expansion of the AIL gene family in apple. The analysis of cis-regulatory elements in MdAIL promoter regions indicated that most of the MdAIL genes are involved in embryo development and seed germination. Moreover, the analysis of tissue-specific expression patterns and transcript levels in adventitious bud regeneration indicated that MdAIL genes play an extensive regulatory role in apple growth and development, especially in the regulation of germination and adventitious bud regeneration from in vitro leaves of apple. In conclusion, this is the first genome-wide analysis of the AIL genes in apple. The current results may help in better understanding the evolution and function of MdAIL genes and thus facilitate further research on plant growth and development.

Differential pulp cell wall structures lead to diverse fruit textures in apple (Malus domestica).

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.

Insertion of a TRIM-like sequence in MdFLS2-1 promoter is associated with its allele-specific expression in response to Alternaria alternata in apple.

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the major fungal diseases in apple. Early field observations revealed, the anther-derived homozygote Hanfu line (HFTH1) was highly susceptible to AAAP, whereas Hanfu (HF) exhibited resistance to AAAP. To understand the molecular mechanisms underlying the difference in sensitivity of HF and HFTH1 to AAAP, we performed allele-specific expression (ASE) analysis and comparative transcriptomic analysis before and after AAAP inoculation. We reported an important immune gene, namely, MdFLS2, which displayed strong ASE in HF with much lower expression levels of HFTH1-derived alleles. Transient overexpression of the dominant allele of MdFLS2-1 from HF in GL-3 apple leaves could enhance resistance to AAAP and induce expression of genes related to salicylic acid pathway. In addition, MdFLS2-1 was identified with an insertion of an 85-bp terminal-repeat retrotransposon in miniature (TRIM) element-like sequence in the upstream region of the nonreference allele. In contrast, only one terminal direct repeat (TDR) from TRIM-like sequence was present in the upstream region of the HFTH1-derived allele MdFLS2-2. Furthermore, the results of luciferase and ß-glucuronidase reporter assays demonstrated that the intact TRIM-like sequence has enhancer activity. This suggested that insertion of the TRIM-like sequence regulates the expression level of the allele of MdFLS2, in turn, affecting the sensitivity of HF and HFTH1 to AAAP.

RNA-Seq profiling reveals the plant hormones and molecular mechanisms stimulating the early ripening in apple.

Fruit development and ripening are essential components of human and animal diets. Fruit ripening is also a vital plant trait for plant shelf life at the commercial level. In the present study, two apple cultivars, Hanfu wild (HC) and Hanfu mutant (HM), were employed for RNA-Sequencing (RNA-Seq) to explore the genes involved in fruit ripening. We retrieved 2642 genes, differentially expressed in HC and HM apple cultivars. Gene ontology (GO) analysis revealed the 569 categories, significantly enriched in biological process, cellular component, and molecular function. KEGG analysis exhibited the plant hormone transduction and flavonoid-anthocyanin biosynthesis pathways, might be involved in the fruit ripening and anthocyanin biosynthesis mechanism. A cluster of 13 and 26 DEGs was retrieved, representing the plant hormones and transcription factors, respectively, that may be important for early ripening in HM genotype. This transcriptome study would be useful for researchers to functionally characterize the DEGs responsible for early ripening.

Application of genome-wide insertion/deletion markers on genetic structure analysis and identity signature of Malus accessions.

BACKGROUND: Apple (Malus ssp.), one of the most important temperate fruit crops, has a long cultivation history and is economically important. To identify the genetic relationships among the apple germplasm accessions, whole-genome structural variants identified between M. domestica cultivars 'Jonathan' and 'Golden Delicious' were used. RESULTS: A total of 25,924 insertions and deletions (InDels) were obtained, from which 102 InDel markers were developed. Using the InDel markers, we found that 942 (75.3%) of the 1251 Malus accessions from 35 species exhibited a unique identity signature due to their distinct genotype combinations. The 102 InDel markers could distinguish 16.7-71.4% of the 331 bud sports derived from 'Fuji', 'Red Delicious', 'Gala', 'Golden Delicious', and other cultivars. Five distinct genetic patterns were found in 1002 diploid accessions based on 78 bi-allele InDel markers. Genetic structure analysis indicated that M. domestica showed higher genetic diversity than the other species. Malus underwent a relatively high level of wild-to-crop or crop-to-wild gene flow. M. sieversii was closely related to both M. domestica and cultivated Chinese cultivars. CONCLUSIONS: The identity signatures of Malus accessions can be used to determine distinctness, uniformity, and stability. The results of this study may also provide better insight into the genetic relationships among Malus species.

Major Latex Protein MdMLP423 Negatively Regulates Defense against Fungal Infections in Apple.

Major latex proteins (MLPs) play critical roles in plants defense and stress responses. However, the roles of MLPs from apple (Malus × domestica) have not been clearly identified. In this study, we focused on the biological role of MdMLP423, which had been previously characterized as a potential pathogenesis-related gene. Phylogenetic analysis and conserved domain analysis indicated that MdMLP423 is a protein with a 'Gly-rich loop' (GXGGXG) domain belonging to the Bet v_1 subfamily. Gene expression profiles showed that MdMLP423 is mainly expressed in flowers. In addition, the expression of MdMLP423 was significantly inhibited by Botryosphaeria berengeriana f. sp. piricola (BB) and Alternaria alternata apple pathotype (AAAP) infections. Apple calli overexpressing MdMLP423 had lower expression of resistance-related genes, and were more sensitive to infection with BB and AAAP compared with non-transgenic calli. RNA-seq analysis of MdMLP423-overexpressing calli and non-transgenic calli indicated that MdMLP423 regulated the expression of a number of differentially expressed genes (DEGs) and transcription factors, including genes involved in phytohormone signaling pathways, cell wall reinforcement, and genes encoding the defense-related proteins, AP2-EREBP, WRKY, MYB, NAC, Zinc finger protein, and ABI3. Taken together, our results demonstrate that MdMLP423 negatively regulates apple resistance to BB and AAAP infections by inhibiting the expression of defense- and stress-related genes and transcription factors.

Genome-wide identification and expression analysis of major latex protein (MLP) family genes in the apple (Malus domestica Borkh.) genome.

Major latex protein/ripening-related proteins (MLP/RRP) subfamily are a class of proteins that play crucial roles in response to defense and stress response. However, their biological function is still not clear, the identification and characterization will provide essential information for understanding their roles. Here, we carried out a genome-wide evolutionary characteristics and gene expression analysis of the MLP family in apple (Malus domestica, Borkh.). A total of 36 MdMLP genes were screened in apple genome. They were uneven located on 5 chromosomes, where were mainly arranged in tandem clusters, and the phylogenetic analysis put forward further views on the evolutionary relationship and putative functions among the genes. The conserved motifs showed that the MLP proteins which contained motif 1 had the potential function, and tissue-specific expression analysis showed that apple MLP members had diverse biological roles. Furthermore, the results showed seven of the MdMLPs that harbored cis-acting regulatory elements in response to defense and stress, and our expression data proved that they were involved in biotic stresses. The present study provides new views to the evolution and regulation of MdMLP genes, which represent objectives of future research and incorporate in resistance-related molecular breeding projects.

An Integrated Transcriptome and Proteome Analysis Reveals New Insights into Russeting of Bagging and Non-Bagging "Golden Delicious" Apple.

Apple skin russeting naturally occurs in many varieties, particularly in "Golden Delicious" and its pedigree, and is regarded as a non-invasive physiological disorder partly caused by excessive deposition of lignin. However, the understanding of its molecular mechanism is still limited. In this study, we used iTRAQ (isobaric tags for relative and absolute quantitation) and RNA-seq to detect the changes in the expression levels of genes and proteins in three developmental stages of russeting formation, in russeted (non-bagging) and non-russeted (bagging) skin of "Golden Delicious" apple. 2856 differentially expressed genes and 942 differentially expressed proteins in the comparison groups were detected at the transcript level and protein level, respectively. A correlation analysis of the transcriptomics and proteomics data revealed that four genes (MD03G1059200, MD08G1009200, MD17G1092400, and MD17G1225100) involved in lignin biosynthesis are significant changed during apple russeting formation. Additionally, 92 transcription factors, including 4 LIM transcription factors, may be involved in apple russeting formation. Among them, one LIM transcription factor (MD15G1068200) was capable of binding to the PAL-box like (CCACTTGAGTAC) element, which indicated it was potentially involved in lignin biosynthesis. This study will provide further views on the molecular mechanisms controlling apple russeting formation.

A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour.

A complete and accurate genome sequence provides a fundamental tool for functional genomics and DNA-informed breeding. Here, we assemble a high-quality genome (contig N50 of 6.99 Mb) of the apple anther-derived homozygous line HFTH1, including 22 telomere sequences, using a combination of PacBio single-molecule real-time (SMRT) sequencing, chromosome conformation capture (Hi-C) sequencing, and optical mapping. In comparison to the Golden Delicious reference genome, we identify 18,047 deletions, 12,101 insertions and 14 large inversions. We reveal that these extensive genomic variations are largely attributable to activity of transposable elements. Interestingly, we find that a long terminal repeat (LTR) retrotransposon insertion upstream of MdMYB1, a core transcriptional activator of anthocyanin biosynthesis, is associated with red-skinned phenotype. This finding provides insights into the molecular mechanisms underlying red fruit coloration, and highlights the utility of this high-quality genome assembly in deciphering agriculturally important trait in apple.

Functional characterization of an apple (Malus x domestica) LysM domain receptor encoding gene for its role in defense response.

Apple gene, MD09G1111800, was identified as a chitin binding receptor-like kinase based on sequence similarity to AtCERK1 (chitin elicitor receptor kinase 1) from Arabidopsis. Sequence analysis on genomic structure, domain composition and transcriptional response to exogenous chitin treatment indicated that MD09G1111800 is an ortholog to AtCERK1 and was therefore named as MdCERK1. Tissue specific expression patterns indicated that MdCERK1 is primarily functional in vegetative tissues of leaf and root, rather than flower, fruit and seed of apple plant. The transcriptional regulation patterns in response to infection by Rhizoctonia solani demonstrated that MdCERK1 is a functional pattern recognition receptor protein (PRR) in apple root tissues. The ability of purified GST-MdCERK1 fusion protein to bind chitin molecules added biochemical evidence for its role in chitin mediated immune responses. An untargeted proteomic approach was also employed for identifying its putative in vivo interaction partners in apple root cells, and results indicated the existence of a functional receptor complex. These data support the conclusion that MdCERK1 is a chitin binding receptor kinase functioning in apple vegetative tissues, which plays an important role in defense activation in response to pathogen infection.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots.

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch Alternaria alternata.

Understanding the defence mechanisms used by apple leaves against Alternaria alternate pathogen infection is important for breeding purposes. To investigate the ultrastructural differences between leaf tissues of susceptible and resistant seedlings, in vitro inoculation assays and transmission electron microscopy (TEM) analysis were conducted with two different inoculation assays. The results indicated that the resistant leaves may have certain antifungal activity against A. alternate that is lacking in susceptible leaves. To elucidate the two different host responses to A. alternate infection in apples, the proteomes of susceptible and resistant apple leaves that had or had not been infected with pathogen were characterised using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). MS identified 43 differentially expressed proteins in two different inoculation assays. The known proteins were categorised into 5 classes, among these proteins, some pathogenesis-related (PR) proteins, such as beta-1,3-glucanase, ascorbate peroxidase (APX), glutathione peroxidase (GPX) and mal d1, were identified in susceptible and resistant hosts and were associated with disease resistance of the apple host. In addition, the different levels of mal d1 in susceptible and resistant hosts may contribute to the outstanding anti-disease properties of resistant leaves against A. alternate. Taken together, the resistance mechanisms of the apple host against A. alternate may be a result of the PR proteins and other defence-related proteins. Given the complexity of the biology involved in the interaction between apple leaves and the A. alternate pathogen, further investigation will yield more valuable insights into the molecular mechanisms of suppression of the A. alternate pathogen. Overall, we outline several novel insights into the response of apple leaves to pathogen attacks. These findings increase our knowledge of pathogen resistance mechanisms, and the data will also promote further investigation into the regulation of the expression of these target proteins.

Genome-wide identification and analysis of the MADS-box gene family in apple.

The MADS-box gene family is one of the most widely studied families in plants and has diverse developmental roles in flower pattern formation, gametophyte cell division and fruit differentiation. Although the genome-wide analysis of this family has been performed in some species, little is known regarding MADS-box genes in apple (Malus domestica). In this study, 146 MADS-box genes were identified in the apple genome and were phylogenetically clustered into six subgroups (MIKC(c), MIKC*, Mα, Mß, Mγ and Mδ) with the MADS-box genes from Arabidopsis and rice. The predicted apple MADS-box genes were distributed across all 17 chromosomes at different densities. Additionally, the MADS-box domain, exon length, gene structure and motif compositions of the apple MADS-box genes were analysed. Moreover, the expression of all of the apple MADS-box genes was analysed in the root, stem, leaf, flower tissues and five stages of fruit development. All of the apple MADS-box genes, with the exception of some genes in each group, were expressed in at least one of the tissues tested, which indicates that the MADS-box genes are involved in various aspects of the physiological and developmental processes of the apple. To the best of our knowledge, this report describes the first genome-wide analysis of the apple MADS-box gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family.

MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.

Anthocyanin and proanthocyanidin (PA) are important secondary metabolites and beneficial to human health. Their biosynthesis is induced by jasmonate (JA) treatment and regulated by MYB transcription factors (TFs). However, which and how MYB TFs regulate this process is largely unknown in apple. In this study, MdMYB9 and MdMYB11 which were induced by methyl jasmonate (MeJA) were functionally characterized. Overexpression of MdMYB9 or MdMYB11 promoted not only anthocyanin but also PA accumulation in apple calluses, and the accumulation was further enhanced by MeJA. Subsequently, yeast two-hybrid, pull-down and bimolecular fluorescence complementation assays showed that both MYB proteins interact with MdbHLH3. Moreover, Jasmonate ZIM-domain (MdJAZ) proteins interact with MdbHLH3. Furthermore, chromatin immunoprecipitation-quantitative PCR and yeast one-hybrid assays demonstrated that both MdMYB9 and MdMYB11 bind to the promoters of ANS, ANR and LAR, whereas MdbHLH3 is recruited to the promoters of MdMYB9 and MdMYB11 and regulates their transcription. In addition, transient expression assays indicated that overexpression of MdJAZ2 inhibits the recruitment of MdbHLH3 to the promoters of MdMYB9 and MdMYB11. Our findings provide new insight into the mechanism of how MeJA regulates anthocyanin and PA accumulation in apple.

The methylation of the PcMYB10 promoter is associated with green-skinned sport in Max Red Bartlett pear.

Varieties of the European pear (Pyrus communis) can produce trees with both red- and green-skinned fruits, such as the Max Red Bartlett (MRB) variety, although little is known about the mechanism behind this differential pigmentation. In this study, we investigated the pigmentation of MRB and its green-skinned sport (MRB-G). The results suggest that a reduction in anthocyanin concentration causes the MRB-G sport. Transcript levels of PcUFGT (for UDP-glucose:flavonoid 3-O-glucosyltransferase), the key structural gene in anthocyanin biosynthesis, paralleled the change of anthocyanin concentration in both MRB and MRB-G fruit. We cloned the PcMYB10 gene, a transcription factor associated with the promoter of PcUFGT. An investigation of the 2-kb region upstream of the ATG translation start site of PcMYB10 showed the regions -604 to -911 bp and -1,218 to -1,649 bp to be highly methylated. A comparison of the PcMYB10 promoter methylation level between the MRB and MRB-G forms indicated a correlation between hypermethylation and the green-skin phenotype. An Agrobacterium tumefaciens infiltration assay was conducted on young MRB fruits by using a plasmid constructed to silence endogenous PcMYB10 via DNA methylation. The infiltrated fruits showed blocked anthocyanin biosynthesis, higher methylation of the PcMYB10 promoter, and lower expression of PcMYB10 and PcUFGT. We suggest that the methylation level of PcMYB10 is associated with the formation of the green-skinned sport in the MRB pear. The potential mechanism behind the regulation of anthocyanin biosynthesis is discussed.