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This article examines the morphological characteristics of apple varieties in northeastern Algeria, specifically focusing on the leaves, fruits, and terminal shoots of three apple varieties: Golden Delicious, Royal Gala, and Starkrimson. The study reveals limited morphological diversity among the investigated apple cultivars, with some descriptors showing insignificant differences between cultivar types. The findings emphasize the distinct physiological and morphological characteristics of each variety and highlight the need for further exploration and conservation efforts. Another document provides information on the application rate of sheep manure and urea for soil salinity levels, as well as the physical and chemical analysis of soil in an orchard. It also discusses the growth habit and length of annual shoots for different apple tree varieties, as well as the flowering characteristics and fruit characteristics such as weight, length, width, shape, color, and ripening period. The document is valuable for understanding the cultivation and quality of apple trees in the studied orchard. Lastly, a document discusses the characterization of new apple cultivars and their implications for sustainable development and consumption. The study found significant variation in vegetative growth, fruit weight, and color among the three cultivated apple tree varieties examined. The authors suggest further research and evaluation to fully understand the genetic diversity and potential uses of these cultivars, emphasizing the importance of rigorous protocols for measurement and observation. The document includes a list of references for further reading on apple diversity and characterization. [Extracted from the article]

The regulation of apple (Malus domestica) fruit texture during ripening is complex and a fundamental determinant of its commercial quality. In climacteric fruit, ripening-related processes are regulated by ethylene (ET), and jasmonate (JA) is also involved in the ethylene biosynthesis pathway, mainly through the transcription factor MYC2. However, the molecular genetic mechanism for fruit ripening processes between the JA and ET signaling pathways still needs to be elucidated. In order to explore how JA regulates apple fruit ripening through ERF4, we used 'Gala' and 'Ralls Janet' fruit at different developmental stages as experimental materials to determine the fruit firmness and related gene expression analysis. Meanwhile, we carried out different hormone treatments on 'Gala' fruit at ripening stage. Here, we show that ERF4 is a core JA signaling hub protein JASMONATE ZIM-DOMAIN (JAZ) interactor that affects ethylene signaling pathways. During fruit development, ERF4 represses the expression of ACS1 and ACO1 by interacting with JAZ, as well as with the JA-activated transcription factor MYC2. Ripening is promoted in JAZ-suppressed apples. Thus, ERF4 acts as a molecular link between ethylene and JA hormone signals, and the natural variation of the ERF4 Ethylene-responsive binding factor-associated amphiphilic repression (EAR) motif decreases repression of ethylene biosynthesis genes. [ABSTRACT FROM AUTHOR]

Societal Impact Statement: The English West Country is the home of cider making, providing the region with jobs and industry, as well as cultural reference points such as Laurie Lee's Cider with Rosie. Many important cider apple varieties were developed at Long Ashton Research Station (LARS), near Bristol, UK, including 29 varieties known collectively as 'The Girls'. After its closure, some of the knowledge and expertise acquired at Long Ashton was lost, including the pedigree of 'The Girls'. We sampled LARS' derived trees and, using a novel genotyping technique, rediscovered the pedigree of 'The Girls', ensuring that this important cider apple collection will be available for future generations. SummaryOur research had two objectives: (a) record the influence of Long Ashton Research Station on the introduction of new cider apple cultivars to the UK; (b) rediscover the parentage of the cider apple cultivars known collectively as 'The Girls'.For rapid, cost effective and accurate genotyping, we used the recently developed, medium density, single nucleotide polymorphism‐based genotyping procedure, SEQSNP®, to characterize the cultivars.We generated a medium density (1,500 markers), whole genome genotype for 245 apple cultivars that allowed us to determine the relationship between cultivars and, in so doing, rediscover the parentage of 'The Girls'.We show that SNP genotyping is an efficient tool for the analysis of genetic diversity in cider apples and apples in general, and that the cider apple breeding programme carried out at Long Ashton Research Station made, and continues to make, a unique contribution to UK cider production. [ABSTRACT FROM AUTHOR]

Background: Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited. Results: Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein–protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of 'Nagafu 2' (CF)/CF than in dwarf trees of CF/'Malling 9' (M.9), and in CF than in the spur-type bud mutation "Yanfu 6" (YF). Conclusion: This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth. [ABSTRACT FROM AUTHOR]

Background: The FK506-binding proteins (FKBPs) play diverse roles in numerous critical processes for plant growth, development, and abiotic stress responses. However, the FKBP gene family in the important fruit crop apple (Malus × domestica Borkh.) has not been studied as thoroughly as in other species. Our research objective was to investigate the mechanisms by which apple FKBPs enable apple plants to tolerate the effects of abiotic stresses. Results: Using bioinformatics-based methods, RT-PCR, and qRT-PCR technologies, we identified 38 FKBP genes and cloned 16 of them in the apple genome. The phylogenetic analysis revealed three major groups within that family. The results from sequence alignments, 3-D structures, phylogenetics, and analyses of conserved domains indicated that apple FKBPs are highly and structurally conserved. Furthermore, genomics structure analysis showed that those genes are also highly and structurally conserved in several other species. Comprehensive qRT-PCR analysis found various expression patterns for MdFKBPs in different tissues and in plant responses to water-deficit and salt stresses. Based on the results from interaction network and co-expression analyses, we determined that the pairing in the MdFKBP62a/MdFKBP65a/b-mediated network is involved in water-deficit and salt-stress signaling, both of which are uniformly up-regulated through interactions with heat shock proteins in apple. Conclusions: These results provide new insight for further study of FKBP genes and their functions in abiotic stress response and multiple metabolic and physiological processes in apple. [ABSTRACT FROM AUTHOR]

Recent studies with Arabidopsis and soybean have shown that a class of valine-glutamine (VQ) motif-containing proteins interacts with some WRKY transcription factors. However, little is known about the evolution, structures, and functions of those proteins in apple. Here, we examined their features and identified 49 apple VQ genes. Our evolutional analysis revealed that the proteins could be clustered into nine groups together with their homologues in 33 species. Historically, the main characteristics of proteins in Groups I, V, VI, VII, IX, and X were thought to have been generated before the monocot-dicot split, whereas those in Groups II, III + IV, and VIII were generated after that split. In the structural analysis, apple MdVQ proteins appeared to bind only with Group I and IIc MdWRKY proteins. Meanwhile, MdVQ1, MdVQ10, MdVQ15, and MdVQ36 interacted with multiple MdVQ proteins to form heterodimers but MdVQ15 formed a homodimer. The functional analysis indicated that overexpression of some apple MdVQ s in Arabidopsis and tobacco plants effected their vegetative and reproductive growth. These results provide important information about the characteristics of apple MdVQ genes and can serve as a solid foundation for further studies about the role of WRKY-VQ interactions in regulating apple developmental and defense mechanisms. [ABSTRACT FROM AUTHOR]

Antioxidant enzymes were well-known for reactive oxygen scavenging and protecting cells from oxidative damage in multiple biotic and abiotic stresses. Aimed to overview of antioxidant enzyme gene families and screen for drought and low-temperature response members, the whole apple antioxidant enzyme genes in apple genome were studied. Out of 73candidates in seven antioxidant enzyme gene families among the entire apple genome, a total of 49 genes were identified, showed more antioxidant enzyme gene members than those in other plant species. A phylogenetic tree based on predicted functional domains and motifs of apple and Arabidopsis antioxidant enzymes revealed 19 putative drought or low-temperature response genes, and 9 out 19 genes belong to five families responded to both drought and low-temperature stresses. Subsequently, cis-elements in those nine antioxidant enzyme gene promoters were examined and abundant elements involved in multiple hormone regulation and abiotic stresses response, including drought and low-temperature were found. The tissue expression specificity for those nine members in eight kinds of tissues was also examined. Screening and identifying both drought and low-temperature response genes provide useful information to understand gene functions and promote application of antioxidant enzyme genes in apple abiotic resistance breeding. [ABSTRACT FROM AUTHOR]

Some horticultural characteristics and bioactive contents of some local apple genotypes grown in Bolu province located in western black sea region were investigated. Along with the horticultural characteristics (fruit weight, width etc.), the phenolic spectrum and organic acids as bioactive content were determined. Among major phenolic compounds, catechin, chlorgenic acid, gallic acid, phloridzin and ratine content varied between 10.84- 5.16 mg 100 ml-1; 25.16-7.17 mg 100 ml-1; 7.35- 1.42 mg 100 ml-1; 6.12-1.18 mg 100 ml-1 and 5.16-1.01 mg 100 ml-1, respectively. The dominant organic acids were malic acid and fumaric acid among the organic acids. When the apple genotypes were evaluated overall in terms of horticultural properties and bioctive contents; it is suggested that 14BL02, 14BL09, 14BL06 and 14BL01 genotypes displayed superior properties and they may create a significant genetic resource to be use them in breeding activities in apple in future. [ABSTRACT FROM AUTHOR]

Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply. [ABSTRACT FROM AUTHOR]

Summary: Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single‐base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought‐sensitive and drought‐tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter‐unmethylated genes showed higher expression levels than promoter‐methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage oxygenases (CCOs) are able to cleave carotenoids to produce apocarotenoids and their derivatives, which are important for plant growth and development. In this study, 21 apple CCO genes were identified and divided into six groups based on their phylogenetic relationships. We further characterized the apple CCO genes in terms of chromosomal distribution, structure and the presence of cis -elements in the promoter. We also predicted the cellular localization of the encoded proteins. An analysis of the synteny within the apple genome revealed that tandem, segmental, and whole-genome duplication events likely contributed to the expansion of the apple carotenoid oxygenase gene family. An additional integrated synteny analysis identified orthologous carotenoid oxygenase genes between apple and Arabidopsis thaliana , which served as references for the functional analysis of the apple CCO genes. The net photosynthetic rate, transpiration rate, and stomatal conductance of leaves decreased, while leaf stomatal density increased under drought and saline conditions. Tissue-specific gene expression analyses revealed diverse spatiotemporal expression patterns. Finally, hormone and abiotic stress treatments indicated that many apple CCO genes are responsive to various phytohormones as well as drought and salinity stresses. The genome-wide identification of apple CCO genes and the analyses of their expression patterns described herein may provide a solid foundation for future studies examining the regulation and functions of this gene family. [ABSTRACT FROM AUTHOR]

Background: The plant-specific gibberellic acid stimulated Arabidopsis (GASA) gene family is critical for plant development. However, little is known about these genes, particularly in fruit tree species. Results: We identified 15 putative Arabidopsis thaliana GASA (AtGASA) and 26 apple GASA (MdGASA) genes. The identified genes were then characterized (e.g., chromosomal location, structure, and evolutionary relationships). All of the identified A. thaliana and apple GASA proteins included a conserved GASA domain and exhibited similar characteristics. Specifically, the MdGASA expression levels in various tissues and organs were analyzed based on an online gene expression profile and by qRT-PCR. These genes were more highly expressed in the leaves, buds, and fruits compared with the seeds, roots, and seedlings. MdGASA genes were also responsive to gibberellic acid (GA3) and abscisic acid treatments. Additionally, transcriptome sequencing results revealed seven potential flowering-related MdGASA genes. We analyzed the expression levels of these genes in response to flowering-related treatments (GA3, 6-benzylaminopurine, and sugar) and in apple varieties that differed in terms of flowering ('Nagafu No. 2' and 'Yanfu No. 6') during the flower induction period. These candidate MdGASA genes exhibited diverse expression patterns. The expression levels of six MdGASA genes were inhibited by GA3, while the expression of one gene was up-regulated. Additionally, there were expression-level differences induced by the 6-benzylaminopurine and sugar treatments during the flower induction stage, as well as in the different flowering varieties. Conclusion: This study represents the first comprehensive investigation of the A. thaliana and apple GASA gene families. Our data may provide useful clues for future studies and may support the hypotheses regarding the role of GASA proteins during the flower induction stage in fruit tree species. [ABSTRACT FROM AUTHOR]

Light plays a central role in regulating both apple plant yield and fruit quality formation; however, the Phytochrome interacting factors (PIFs), which are the main components of Phytochrome-mediated light signal transduction in apple, have rarely been characterized. Here, we isolated and identified a PIF-like protein(MdPIF1) in apple, which is similar to AtPIF1. MdPIF1 was constitutively expressed at different levels in various apple tissues, and the transcription level of MdPIF1 was significantly induced during seed germination. A functional complementation assay in the Arabidopsis PIF1 -deletion mutant pil5 suggested that MdPIF1 was a negative regulator in the Phy-mediated inhibition of hypocotyl elongation under far-red light conditions. MdPIF1 -overexpression promoted hypocotyl elongation, while inhibiting seed germination and PIF1 deletion-induced the bleaching phenotype in the pil5 mutant. In addition, expression analysis indicated that MdPIF1 was involved in the germination of apple seeds and dormancy breaking of apple buds. Moreover, MdPIF1 inhibited the growth of apple calli via Phy-mediated pathways. These findings build a solid foundation for studies on Phytochrome-mediated light signal transduction and molecular breeding in apple. [ABSTRACT FROM AUTHOR]

RCD1 is a member of the plant-specific SRO protein family. Several SRO genes have been functionally identified in the regulation of abiotic stresses in Arabidopsis and other plant species. However, the function of SROs is largely unknown in apple ( Malus× domestica). In this study, six MdSRO-encoding genes were isolated, categorized into two types and mapped to six chromosomes. The phylogenetic analysis demonstrated that the sequences of the AtSRO and MdSRO proteins are highly conserved. Subsequently, expression analysis showed that MdSRO genes had different expression profiles in different tissues and in response to various stresses. Finally, MdRCD1 was isolated for functional identification. The results showed that resistance to oxidation stress in apple calli was enhanced by MdRCD1 overexpression and weakened by MdRCD1 suppression. MdRCD1 also played a crucial role in the regulation of ROS homeostasis in transgenic apple calli and Arabidopsis. Ectopic expression of MdRCD1 significantly enhanced resistance to salt and oxidative stresses in transgenic lines. In addition, MdRCD1 also enhanced drought tolerance due to its influence on stomatal opening. Based on these results, we conclude that MdRCD1 is an important regulator in abiotic stress response. [ABSTRACT FROM AUTHOR]

Plant GATA transcription factors are type-IV zinc-finger proteins that play important regulatory roles in plant growth and development. In this study, we identified 35 GATA genes classified into four groups in the whole genome sequence of Malus domestica . A physiochemical property analysis indicated that GATA proteins are largely unstable hydrophilic proteins. An analysis of conserved protein motifs uncovered three highly conserved motifs, in addition to the GATA motif, in all MdGATA proteins. These three motifs, CCT, TIFY, and ASXH, were found to occur in specific GATA groups and may be related to GATA gene function. We identified 10 pairs of putative paralogs, indicating that MdGATA genes have mainly undergone whole genome duplication. Eighteen orthologous gene pairs were also identified between Arabidopsis thaliana and M. domestica . Furthermore, many light-responsive cis -elements were found in MdGATA gene promoters. Tissue-specific expression analysis performed by quantitative real-time reverse transcription PCR showed that MdGATA genes were preferentially expressed in flowers, leaves, and buds. Apple seedlings maintained in darkness for 7 days exhibited a moderate decline in chlorophyll content along with significant down-regulation of most MdGATA genes, suggesting that MdGATA genes may be involved in light-responsive development and chlorophyll-level regulation. The distinctly higher expression levels observed for many MdGATA genes during three stages of floral induction also indicate that MdGATA genes may play a role in the apple flowering transition. The results presented here lay the foundation for further investigation of MdGATA gene family putative functions and improvement of apple yields. [ABSTRACT FROM AUTHOR]

Plant growth regulators such as α-naphthylacetic acid (NAA) or 6-benzyladenine (BA) are commonly used for thinning apple fruits. NAs exhibits an auxin- and gibberellin-type biological activity, stimulate the uptake and translocation of auxins in plant tissue, as well as IAA synthesis and act as surfactants. It was assumed that NAs applied at the stadium of early fruit growth might cause fruit abscission in apple, similarly to NAA. The goal of the research was to test the presumed thinning effect of naphthenic acids on apple cultivars 'Golden Delicious' and 'Red Delicious'. Three treatments were applied on both apple cultivars including 16.5 μL L NAA, 200 μL L BA and 2.63 mg L potassium salts of NAs. Cortical cell size and the number of cell layers were measured in order to determine the contribution of cell size and cell division in early fruit growth, as affected by chemical thinning treatments. The effects of plant growth regulators NAA, BA and NAs on fruit set, fruit size and the anatomy of the fruit cortex highly depend on varietal differences. In 'Golden Delicious' fruit set reduction by chemical thinning increases both the number of cell layers and cell size of apple fruit, while in 'Red Delicious' NAA inhibits fruit growth, while BA is ineffective. Cell growth promoting activity of NAA and NAs, accompanied by the decrease in fruit set and reduced competition among fruits within the canopy, results in a significant fruit weight increase at harvest. The research confirms the auxinic properties of NAs, and their assumed thinning activity. [ABSTRACT FROM AUTHOR]

Flavonoids are low-molecular-weight phenolic compounds that are widely distributed in the plant kingdom. They have different roles in plant resistance to biotic and abiotic stresses. The transcription factor gene MdMyb10 (Gene Bank: DQ267896) was introduced into two apple ( Malus domestica Borkh.) cultivars i.e. ‘Holsteiner Cox (HC)’ and ‘Gala’ via Agrobacterium -mediated transformation. The regenerated shoots were selected on kanamycin containing media. The presence of additional MdMyb10 gene in putative shoots was confirmed by PCR, RT-PCR and Southern blotting. Expression level of introduced MdMyb10 gene was analyzed by quantitative real time PCR. The results confirmed a dramatic increase in overexpression of MdMyb10 in the transgenic plants, up to 1261 and 847-folds for cultivars Holsteiner Cox and Gala, respectively compared to non-transformed negative control plants. HPLC-MS was used to determine the levels of different flavonoid compounds in both non-transgenic and transgenic plants. In MdMyb10 ‘HC’ transgenic plants, some of the polyphenols analyzed were enhanced while others were reduced in comparison to their levels in the non-transgenic plants. On the other hand, all of the analyzed polyphenol classes were induced in MdMyb10 ‘Gala’ transgenic plants in comparison to their levels in the non-transgenic plants. In the present study, the flavonoid pathway was successfully modified in apple by overexpressing the MdMyb10 transcription factor to validate the hypothesis of increased effect on plant disease resistance. [ABSTRACT FROM AUTHOR]

GRAS genes encode plant-specific transcription factors that play important roles in plant growth and development. However, little is known about the GRAS gene family in apple. In this study, 127 GRAS genes were identified in the apple (Malus domestica Borkh.) genome and named MdGRAS1 to MdGRAS127 according to their chromosomal locations. The chemical characteristics, gene structures and evolutionary relationships of the MdGRAS genes were investigated. The 127 MdGRAS genes could be grouped into eight subfamilies based on their structural features and phylogenetic relationships. Further analysis of gene structures, segmental and tandem duplication, gene phylogeny and tissue-specific expression with ArrayExpress database indicated their diversification in quantity, structure and function. We further examined the expression pattern of MdGRAS genes during apple flower induction with transcriptome sequencing. Eight higher MdGRAS (MdGRAS6, 26, 28, 44, 53, 64, 107, and 122) genes were surfaced. Further quantitative reverse transcription PCR indicated that the candidate eight genes showed distinct expression patterns among different tissues (leaves, stems, flowers, buds, and fruits). The transcription levels of eight genes were also investigated with various flowering related treatments (GA3, 6-BA, and sucrose) and different flowering varieties (Yanfu No. 6 and Nagafu No. 2). They all were affected by flowering-related circumstance and showed different expression level. Changes in response to these hormone or sugar related treatments indicated their potential involvement during apple flower induction. Taken together, our results provide rich resources for studying GRAS genes and their potential clues in genetic improvement of apple flowering, which enriches biological theories of GRAS genes in apple and their involvement in flower induction of fruit trees. [ABSTRACT FROM AUTHOR]

The bHLH (basic helix-loop-helix) transcription factor family is the second largest in plants. It occurs in all three eukaryotic kingdoms, and plays important roles in regulating growth and development. However, family members have not previously been studied in apple. Here, we identified 188 MdbHLH proteins in apple "Golden Delicious" (Malus × domestica Borkh.), which could be classified into 18 groups. We also investigated the gene structures and 12 conserved motifs in these MdbHLHs. Coupled with expression analysis and protein interaction network prediction, we identified several genes thatmight be responsible for abiotic stress responses. This study provides insight and rich resources for subsequent investigations of such proteins in apple. [ABSTRACT FROM AUTHOR]

The annual growth cycle of trees is the result of seasonal cues. The onset of winter triggers an endodormant state preventing bud growth and, once a chilling requirement is satisfied, these buds enter an ecodormant state and resume growing. MADS-box genes with similarity to Arabidopsis SHORT VEGETATIVE PHASE (SVP) [the SVP-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes] have been implicated in regulating flowering and growth-dormancy cycles in perennials. Here, we identified and characterized three DAM-like (MdDAMs) and two SHORT VEGETATIVE PHASElike (MdSVPs) genes from apple (Malus x domestica 'Royal Gala'). The expression of MdDAMa and MdDAMc indicated they may play a role in triggering autumn growth cessation. In contrast, the expression of MdDAMb, MdSVPa and MdSVPb suggested a role in maintaining bud dormancy. Consistent with this, ectopic expression of MdDAMb and MdSVPa in 'Royal Gala' apple plants resulted in delayed budbreak and architecture change due to constrained lateral shoot outgrowth, but normal flower and fruit development. The association of MdSVPa and MdSVPb expression with floral bud development in the low fruiting 'Off' trees of a biennial bearing cultivar 'Sciros' suggested the SVP genes might also play a role in floral meristem identity. [ABSTRACT FROM AUTHOR]

Elemental phosphorus (Pi) is essential to plant growth and development. The family of phosphate transporters (PHTs) mediates the uptake and translocation of Pi inside the plants. Members include five sub-cellular phosphate transporters that play different roles in Pi uptake and transport. We searched the Genome Database for Rosaceae and identified five clusters of phosphate transporters in apple (Malus domestica), including 37 putative genes. The MdPHT1 family contains 14 genes while MdPHT2 has two, MdPHT3 has seven, MdPHT4 has 11, and MdPHT5 has three. Our overview of this gene family focused on structure, chromosomal distribution and localization, phylogenies, andmotifs. These genes displayed differential expression patterns in various tissues. For example, expression was high for MdPHT1;12, MdPHT3;6, and MdPHT3;7 in the roots, and was also increased in response to low-phosphorus conditions. In contrast, MdPHT4;1, MdPHT4;4, and MdPHT4;10 were expressed only in the leaves while transcript levels of MdPHT1;4, MdPHT1;12, and MdPHT5;3 were highest in flowers. In general, these 37 genes were regulated significantly in either roots or leaves in response to the imposition of phosphorus and/or drought stress. The results suggest that members of the PHT family function in plant adaptations to adverse growing environments. Our study will lay a foundation for better understanding the PHT family evolution and exploring genes of interest for genetic improvement in apple. [ABSTRACT FROM AUTHOR]

Key message : Here, we report the decrease of JA-sensitivity and enhancement of tolerance to salt and PEG stresses in Arabidopsis overexpressing apple MdJAZ2. Abstract: As signalling molecules, jasmonates (JAs) play significant roles in plant development and stress responses. JAZ proteins are the targets of the SCF complex and act as the negative regulators in JA signalling pathway. However, there are no reports regarding the biological function of apple JAZ genes. In this study, one JAZ gene, MdJAZ2 from apple, was functionally characterized in detail. The expression of MdJAZ2 was up-regulated by MeJA and wounding treatments. MdJAZ2-GFP fusion protein was observed in nucleus in transient expression assay. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that MdJAZ2 could form homo- and heteromers, and also interact with F-box protein MdCOI1. Overexpression of MdJAZ2 conferred impaired JA-sensitivity in transgenic Arabidopsis, including JA-mediated root growth inhibition, susceptibility to the bacterial pathogen Pst DC3000, and the expression of JA response genes. Additionally, MdJAZ2 overexpression also improved tolerance to NaCl and PEG treatments in transgenic Arabidopsis. Together, our findings suggest that apple MdJAZ2 was not only involved in the JA response but also played roles in stress tolerance. [ABSTRACT FROM AUTHOR]

Ripening in climacteric fruit requires the gaseous phytohormone ethylene. Although ethylene signaling has been well studied, knowledge of the transcriptional regulation of ethylene biosynthesis is still limited. Here we show that an apple ( Malus domestica) ethylene response factor, Md ERF2, negatively affects ethylene biosynthesis and fruit ripening by suppressing the transcription of Md ACS1, a gene that is critical for biosynthesis of ripening-related ethylene. Expression of Md ERF2 was suppressed by ethylene during ripening of apple fruit, and we observed that Md ERF2 bound to the promoter of Md ACS1 and directly suppressed its transcription. Moreover, Md ERF2 suppressed the activity of the promoter of Md ERF3, a transcription factor that we found to bind to the Md ACS1 promoter, thereby increasing Md ACS1 transcription. We determined that the Md ERF2 and Md ERF3 proteins directly interact, and this interaction suppresses the binding of Md ERF3 to the Md ACS1 promoter. Moreover, apple fruit with transiently downregulated Md ERF2 expression showed higher ethylene production and faster ripening. Our results indicate that Md ERF2 negatively affects ethylene biosynthesis and fruit ripening in apple by suppressing the transcription of Md ACS1 via multiple mechanisms, thereby acting as an antagonist of positive ripening regulators. Our findings offer a deep understanding of the transcriptional regulation of ethylene biosynthesis during climacteric fruit ripening. [ABSTRACT FROM AUTHOR]

Tubby-like proteins (TLPs), which have a highly conserved b barrel tubby domain, have been found to be associated with some animal-specific characteristics. In the plant kingdom, more than 10 TLP family members were identified in Arabidopsis, rice and maize, and they were found to be involved in responses to stress. The publication of the apple genome makes it feasible to systematically study the TLP family in apple. In this investigation, nine TLP encoding genes (TLPs for short) were identified. When combined with the TLPs from other plant species, the TLPs were divided into three groups (group A, B, and C). Most plant TLP members in group A contained an additional F-box domain at the N-terminus. However, no common domain was identified other than tubby domain either in group B or in group C. An analysis of the tubby domains of MdTLPs identified three types of conserved motifs. Motif 1 and 2, the signature motifs in the confirmed TLPs, were always present in MdTLPs, while motif 3 was absent from group B. Homology modeling indicated that the tubby domain of most MdTLPs had a closed β barrel, as in animal tubby domains. Expression profiling revealed that the MdTLP genes were expressed in multiple organs and were abundant in roots, stems, and leaves but low in flowers. An analysis of cis-acting elements showed that elements related to the stress response were prevalent in the promoter sequences of MdTLPs. Expression profiling by qRT-PCR indicated that almost all MdTLPs were up-regulated at some extent under abiotic stress, exogenous ABA and H2O2 treatments in leaves and roots, though different MdTLP members exhibited differently in leaves and roots. The results and information above may provide a basis for further investigation of TLP function in plants. [ABSTRACT FROM AUTHOR]

In this study, we characterized a naturally occurring self-compatible apple cultivar, ‘CAU-1’ (S 1 S 9 ), and studied the underlying mechanism that causes its compatibility. Analyses of both fruit set rate and seed number after self-pollination or cross-pollination with ‘Fuji’ (S 1 S 9 ), and of pollen tube growth, demonstrated that ‘CAU-1’ is self-compatible. Genetic analysis by S-RNase PCR-typing of selfed progeny of ‘CAU-1’ revealed the presence of all progeny classes (S 1 S 1 , S 1 S 9 , and S 9 S 9 ). Moreover, no evidence of S-allele duplication was found. These findings support the hypothesis that loss of function of an S-locus unlinked pollen-part mutation (PPM) expressed in pollen, rather than a natural mutation in the pollen-S gene (S 1 - and S 9 - haplotype), leads to SI breakdown in ‘CAU-1’. In addition, there were no significant differences in pollen morphology or fertility between ‘Fuji’ and ‘CAU-1’. However, we found that the effect of S 1 - and S 9 -RNase on the SI behavior of pollen could not be addressed better in ‘CAU-1’ than in ‘Fuji’. Furthermore, we found that a pollen-expressed hexose transporter, MdHT1, interacted with S-RNases and showed significantly less expression in ‘CAU-1’ than in ‘Fuji’ pollen tubes. These findings support the hypothesis that MdHT1 may participate in S-RNase internalization during the SI process, and decrease of MdHT1 expression in ‘CAU-1’ hindered the release of self S-RNase into the cytoplasm of pollen tubes, thereby protecting pollen from the cytotoxicity of S-RNase, finally probably resulting in self-compatibility. Together, these findings indicate that S-locus external factors are required for gametophytic SI in the Rosaceae subtribe Pyrinae . [ABSTRACT FROM AUTHOR]

UVR8 (UV Resistance Locus 8) is an ultraviolet-B (UV-B; 280–315 nm) light receptor that is involved in regulating many aspects of plant growth and development. UV-B irradiation can increase the development of flower and fruit coloration in many fruit trees, such as grape, pear and apple. Previous investigations of the structure and functions of UVR8 in plants have largely focused on Arabidopsis. Here, we isolated the UVR8 gene from apple ( Malus domestica ) and analyzed its function in transgenic Arabidopsis. Genomic and protein sequence analysis showed that MdUVR8 shares high similarity with the AtUVR8 protein from Arabidopsis, including the conserved seven-bladed β-propeller, the C27 region, the 3 “GWRHT” motifs and crucial amino-acid residues (14 Trps, 2 Args). A point mutation prediction and three-dimensional structural analysis of MdUVR8 indicated that it has a similar structure to AtUVR8 and that the crucial residues are also important in MdUVR8. In terms of transcript levels, MdUVR8 expression was up-regulated by UV-B light, which suggests that its expression follows a 24-h circadian rhythm. Using heterologous expression of MdUVR8 in both uvr8-1 mutant and wild-type (WT) Arabidopsis, we found that MdUVR8 regulates hypocotyl elongation and gene expression under UV-B light. These data provide functional evidence for a role of MdUVR8 in controlling photomorphogenesis under UV-B light and indicate that the function of UVR8 is conserved between Arabidopsis and apple. Furthermore, we examined the interaction between MdUVR8 and MdCOP1 (constitutive photomorphogenic1) using a yeast two-hybrid assay and a co-immunoprecipitation assay. This interaction provides a direction for investigating the regulatory mechanisms of the UV-B-light pathway in apple. [ABSTRACT FROM AUTHOR]

As one of the largest transcriptional factor families in plants, WRKY genes play significant roles in various biotic and abiotic stress responses. Although the WRKY gene family has been characterized in a few plant species, the details remain largely unknown in the apple ( Malus domestica Borkh.). In this study, we identified a total of 127 MdWRKYs from the apple genome, which were divided into four subgroups according to the WRKY domains and zinc finger motif. Most of them were mapped onto the apple's 17 chromosomes and were expressed in more than one tissue, including shoot tips, mature leaves, fruit and apple calli. We then contrasted WRKY expression patterns between calli grown in solid medium (control) and liquid medium (representing waterlogging stress) and found that 34 WRKY genes were differentially expressed between the two growing conditions. Finally, we determined the expression patterns of 10 selected WRKY genes in an apple rootstock, G41, in response to waterlogging and drought stress, which identified candidate genes involved in responses to water stress for functional analysis. Our data provide interesting candidate MdWRKYs for future functional analysis and demonstrate that apple callus is a useful system for characterizing gene expression and function in apple. [ABSTRACT FROM AUTHOR]

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The ubiquitin-protein ligases (E3s) directly participate in ubiquitin (Ub) transferring to the target proteins in the ubiquitination pathway. The HECT ubiquitin-protein ligase (UPL), one type of E3s, is characterized as containing a conserved HECT domain of approximately 350 amino acids in the C terminus. Some UPLs were found to be involved in trichome development and leaf senescence in Arabidopsis. However, studies on plant UPLs, such as characteristics of the protein structure, predicted functional motifs of the HECT domain, and the regulatory expression of UPLs have all been limited. Here, we present genome-wide identification of the genes encoding UPLs (HECT gene) in apple. The 13 genes (named as MdUPL1- MdUPL13) from ten different chromosomes were divided into four groups by phylogenetic analysis. Among these groups, the encoding genes in the intron-exon structure and the included additional functional domains were quite different. Notably, the F-box domain was first found in MdUPL7 in plant UPLs. The HECT domain in different MdUPL groups also presented different spatial features and three types of conservative motifs were identified. The promoters of each MdUPL member carried multiple stress-response related elements by cis-acting element analysis. Experimental results demonstrated that the expressions of several MdUPLs were quite sensitive to cold-, drought-, and salt-stresses by qRT-PCR assay. The results of this study helped to elucidate the functions of HECT proteins, especially in Rosaceae plants. [ABSTRACT FROM AUTHOR]

Apple breeding is usually done to produce higher quality apples. We crossed two late ripening high quality apple cultivars (i.e., Golden Smoothie and Red Spur) as maternal parents with two Iranian domestic early ripening apple cultivars (i.e., Golab-e Kohanz and Shafi Abadi) as pollen parents in 2005 to breed new early ripening apple cultivars. We then measured qualitative and quantitative traits of 360 off spring during 2010 and 2011. Results of recorded traits on the 5th and 6th year of growth of the progeny showed that during two evaluation years, flowering and fruit ripening time, titrable acidity, SSC/acid ratio, and fruit weight had the highest coefficients of phenotypic variations. Progenies were classified in three groups: early, mid, and late ripening. Some of the selected progeny were better than their early ripening parental cultivars being earlier in the ripening, with larger fruit size and better texture and flavor. Genotypes A31, A32, I5, I23, and I57 were in the early ripening category, B11 and A34 were in the middle, and B77 and E21 were in the late ripening categories. These characteristics were considered to present an improvement. [ABSTRACT FROM PUBLISHER]

The paper deals with the usefulness of Discriminant and Principal Component analyses for determining the relative contribution of morphological and reproductive characters responsible in increasing the yield of apple. The technique Discriminant analysis was applied to formulate categorization rule for allocating the apple tree to 'high' and 'low' yielder groups. This Discriminant equation revealed that the characters canopy spread (X2), Fruit set (X7), LD Ratio (X9) and Fruit weight (X10) are the most important characters that discriminated the two groups. The Principal Component Analysis was extracted for the assessment of relative contribution of morphological and reproductive characters responsible in increasing the yield of apple. In case of high yielders, three of the ten Principal Components (PCs) have Eigen values greater than unity (Gutman's lower bound) which played the main role in the analysis. These components were vegetative characteristics, Plant vigour and flowering characteristics and Fruiting characteristics which explained 33.29%, 22.24% and 13.55% respectively and collectively 69.08% of the total variation of the original variables. In case of low yielders, three principal components had been retained for the analysis. These components were Plant Vigour, Yield and Flowering Characteristics and Fruiting characteristics which explained 35.68%, 22.22% and 12.09% respectively and in aggregate, 69.99% of the total variation of original variables. [ABSTRACT FROM AUTHOR]

Fruit quality is a combination of several features impacting the general consumers' appreciation, and it is thought as the final result of a complex physiological mechanism ongoing during the entire ontogenic life cycle. In the horticultural management apples are normally stored for a long period, ensuring the availability of fresh fruit on the market over a year-round period. In this context, the role of postharvest is to preserve the properties gained at harvest without sacrificing the general fruit quality. In this scenario, the choice of the most appropriate harvest time plays a crucial role, representing a compromise between the achievement of a sufficient quality and the mechanical resistance to harvest and postharvest practice. So far the harvesting time has been defined with too elementary and empirical tools, making thus difficult the selection of a proper harvesting window. In this work, the expression profile of thirteen genes involved in auxin, ethylene as well as cell wall metabolism have been assessed over several dates of two apple cultivars distinguished by a different ripening behavior, such as 'Golden Delicious' and 'Fuji'. The monitoring of the apple fruit maturity by candidate gene transcription profiling propose these elements as novel biomarkers, suggesting the first 10 days, after the commercial harvest, as the most appropriate harvesting window for 'Golden Delicious'. In 'Fuji', instead, the time frame resulted more extended, due to a reduced expression of marker genes and slower ripening progression. [ABSTRACT FROM AUTHOR]

Key message: Salt-induced phosphorylation of MdVHA-B1 protein was mediated by MdSOS2L1 protein kinase, and thereby increasing malate content in apple. Abstract: Salinity is an important environmental factor that influences malate accumulation in apple. However, the molecular mechanism by which salinity regulates this process is poorly understood. In this work, we found that MdSOS2L1, a novel AtSOS2-LIKE protein kinase, interacts with V-ATPase subunit MdVHA-B1. Furthermore, MdSOS2L1 directly phosphorylates MdVHA-B1 at Ser site to modulate malate accumulation in response to salt stress. Meanwhile, a series of transgenic analyses in apple calli showed that the MdSOS2L1-MdVHAB1 pathway was involved in the regulation of malate accumulation. Finally, a viral vector-based transformation approach demonstrated that the MdSOS2L1-MdVHAB1 pathway also modulated malate accumulation in apple fruits with or without salt stress. Collectively, our findings provide a new insight into the mechanism by which MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple. [ABSTRACT FROM AUTHOR]

Bagged fruits of green apple cultivar "Granny Smith" have been found to turn cardinal red after debagging during fruit-ripening in the Loess Plateau region of China. To understand this phenomenon at post-transcriptional level, we have investigated the roles of microRNAs (miRNAs) in response to debagging. Three small RNA libraries were primarily constructed from peels of "Granny Smith" apples subjected to bagging followed by sunlight re-exposure treatments (0, 6 h, 1 day) (debagging), and from peels of apples without any bagging treatments (0, 6 h, 1 day). 201 known miRNAs belonging to 43 miRNA families and 220 novel miRNAs were identified via high-throughput sequencing. Some miRNAs were found to be differentially expressed after debagging, which indicated that miRNAs affected anthocyanin accumulation through their target genes in mature apple. To further explore the effect of debagging on miRNAs regulating the expression of anthocyanin regulatory genes, four miRNAs and their target genes regulating anthocyanin accumulation, miR156, miR828, miR858, and miR5072, were compared between green cultivar "Granny Smith" and red cultivar "Starkrimson." Results showed that mdm-miR828 and mdm-miR858 regulated anthocyanin contents in both apple cultivars, while mdm-miR156 only affected anthocyanin accumulation in "Granny Smith," and miR5072 affected anthocyanin accumulation in "Starkrimson." Additional analysis of gene ontology for the differentially expressed miRNAs after debagging treatments and their predicted target genes showed that they were involved in photo-protective response after debagging from 0 h to 1 day; they might play important roles in fruit development and adaptation to high light stress. [ABSTRACT FROM AUTHOR]

Nucleobase ascorbate transporters (NATs), also known as nucleobase:cation-symporter 2 (NCS2) proteins, belong to an evolutionarily widespread family of transport proteins. Because little is known about NAT genes in apple ( Malus domestica ), this papper screened GDR genome databases and identified 14 NAT s in that species. Here, presented an overview of this gene family in apple, including structures, chromosome distribution and localization, phylogenies, and motifs. Phylogenetic and structure analyses showed that these genes could be assigned to three clusters. The 14 genes selected here displayed differential expression patterns in various tissues. Expression was very high for MdNAT12 , MdNAT4 , and MdNAT6 in the shoot tips, mature leaves, and young and mature fruits. MdNAT1 and MdNAT7 tended to show increased expression while the fruits were maturing. MdNAT6 proved responsive to both drought and salinity. Their transcript levels were generally up-regulated significantly during the fruit maturation process as well as in response to the imposition of drought or salt stress. Results suggest that members of this family function in tissue development, fruit maturation, and plant adaptations to adverse growing environments. [ABSTRACT FROM AUTHOR]

Using RNA-seq, this study analysed an apple (Malusxdomestica) anthocyanin-deficient yellow-skin somatic mutant 'Blondee' (BLO) and its red-skin parent 'Kidd's D-8' (KID), the original name of 'Gala', to understand the molecular mechanisms underlying the mutation. A total of 3299 differentially expressed genes (DEGs) were identified between BLO and KID at four developmental stages and/or between two adjacent stages within BLO and/or KID. A weighted gene co-expression network analysis (WGCNA) of the DEGs uncovered a network module of 34 genes highly correlated (r=0.95, P=9.0 x 10-13) with anthocyanin contents. Although 12 of the 34 genes in the WGCNA module were characterized and known of roles in anthocyanin, the remainder 22 appear to be novel. Examining the expression of ten representative genes in the module in 14 diverse apples revealed that at least eight were significantly correlated with anthocyanin variation. MdMYB10 (MDP0000259614) and MdGST (MDP0000252292) were among the most suppressed module member genes in BLO despite being undistinguishable in their corresponding sequences between BLO and KID. Methylation assay of MdMYB10 and MdGST in fruit skin revealed that two regions (MR3 and MR7) in the MdMYB10 promoter exhibited remarkable differences between BLO and KID. In particular, methylation was high and progressively increased alongside fruit development in BLO while was correspondingly low and constant in KID. The methylation levels in both MR3 and MR7 were negatively correlated with anthocyanin content as well as the expression of MdMYB10 and MdGST. Clearly, the collective repression of the 34 genes explains the loss-of-colour in BLO while the methylation in MdMYB10 promoter is likely causal for the mutation. [ABSTRACT FROM AUTHOR]

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The outer epidermal layer of apple fruit is covered by a protective cuticle. Composed of a polymerized cutin matrix embedded with waxes, the cuticle is a natural waterproof barrier and protects against several abiotic and biotic stresses. In terms of apple production, the cuticle is essential to maintain long post-harvest storage, while severe failure of the cuticle can result in the formation of a disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. This is typically an undesirable consumer trait, and negatively impacts the post-harvest storage of apples. In order to identify genetic factors controlling cuticle biosynthesis (and thus preventing russet) in apple, a quantitative trait locus (QTL) mapping survey was performed on a full-sib population. Two genomic regions located on chromosomes 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high-throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by quantitative real-time PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor gene (termed MdSHN3) on chromosome 15. Orthologues of SHN1/WIN1 have been previously shown to regulate cuticle formation in Arabidopsis, tomato, and barley. The MdSHN3 transcription factor gene displayed extremely low expression in lines with improper cuticle formation, suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit. [ABSTRACT FROM AUTHOR]

Background: Genetic map based quantitative trait locus (QTL) analysis is an important method for studying important horticultural traits in apple. To facilitate molecular breeding studies of fruit quality traits in apple, we aim to construct a high density map which was efficient for QTL mapping and possible to search for candidate genes directly in mapped QTLs regions. Methods: A total of 1733 F1 seedlings derived from 'Jonathan' × 'Golden Delicious' was used for the map constructionand QTL analysis. The SNP markers were developed by restriction site-associated DNA sequencing (RADseq). Phenotyping data of fruit quality traits were calculated in 2008-2011. Once QTLs were mapped, candidate genes were searched for in the corresponding regions of the apple genome sequence underlying the QTLs. Then some of the candidate genes were validated using real-time PCR. Results: A high-density genetic map with 3441 SNP markers from 297 individuals was generated. Of the 3441 markers, 2017 were mapped to 'Jonathan' with a length of 1343.4 cM and the average distance between markers was 0.67 cM, 1932 were mapped to 'Golden Delicious' with a length of 1516.0 cM and the average distance between markers was 0.78 cM. Twelve significant QTLs linked to the control of fruit weight, fruit firmness, sugar content and fruit acidity were mapped to seven linkage groups. Based on gene annotation, 80, 64 and 17 genes related to fruit weight, fruit firmness and fruit acidity, respectively, were analyzed.Among the 17 candidate genes associated with control of fruit acidity, changes in the expression of MDP0000582174 (MdMYB4) were in agreement with the pattern of changes in malic acid content in apple during ripening, and the relative expression of MDP0000239624 (MdME) was significantly correlated withfruit acidity. Conclusions: We demonstrated the construction of a dense SNP genetic map in apple using next generation sequencing and that the increased resolution enabled the detection of narrow interval QTLs linked to the three fruit quality traits assessed. The candidate genes MDP0000582174 and MDP0000239624 were found to be related to fruit acidity regulation. We conclude that application of RADseq for genetic map construction improved the precision of QTL detection and should be utilized in future studies on the regulatory mechanisms of important fruit traits in apple. [ABSTRACT FROM AUTHOR]

Ethylene biosynthesis in plants involves different 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes. The regulation of each ACS gene during fruit development is unclear. Here, we characterized another apple (Malus_domestica) ACS gene, MdACS6. The transcript of MdACS6 was observed not only in fruits but also in other tissues. During fruit development, MdACS6 was initiated at a much earlier stage, whereas MdACS3a and MdACS1 began to be expressed at 35 d before harvest and immediateley after harvest, respectively. Moreover, the enzyme activity of MdACS6 was significantly lower than that of MdACS3a and MdACS1, accounting for the low ethylene biosynthesis in young fruits. Overexpression of MdACS6 (MdACS6-OE) by transient assay in apple showed enhanced ethylene production, and MdACS3a was induced in MdACS6-OE fruits but not in control fruits. In MdACS6 apple fruits silenced by the virus-induced gene silencing (VIGS) system (MdACS6-AN), neither ethylene production nor MdACS3a transcript was detectable. In order to explore the mechanism through which MdACS3a was induced in MdACS6-OE fruits, we investigated the expression of apple ethylene-responsive factor (ERF) genes. The results showed that the expression of MdERF2 was induced in MdACS6-OE fruits and inhibited in MdACS6-AN fruits. Yeast one-hybrid assay showed that MdERF2 protein could bind to the promoter of MdACS3a. Moreover, down-regulation of MdERF2 in apple flesh callus led to a decrease of MdACS3a expression, demonstrating the regulation of MdERF2 on MdACS3a. The mechanism through which MdACS6 regulates the action of MdACS3a was discussed. [ABSTRACT FROM AUTHOR]

Almost all commercially grown apple cultivars in Turkey are exotic, with local varieties serving as important genetic resources for breeding and marker development purposes. Among the latter is Amasya, which has a pleasing taste and flavor, but is unsuitable for commercial use because of its small fruit size and alternate-bearing characteristics. Genetic maps of apple cultivars are important resources to facilitate marker-assisted selection. Because of its good eating quality, Amasya is a good genetic resource for marker development to enhance fruit quality, especially flavor. In this study, we constructed a simple sequence repeat (SSR) marker-based genetic map of the Amasya apple cultivar using 96 F1 progenies generated from a cross between Kaşel-41 (a selection from Amasya) and Williams' Pride cultivars. Of 187 bacterial artificial chromosome SSRs, 150 expressed sequence tag SSRs, and of 362 genomic SSR primer pairs, 400 markers were mapped, with parental and consensus maps constructed independently. A reference map was also constructed using 207 common SSR markers, thereby producing the most dense SSR-based reference map currently reported for apple. The reference genetic map was 1351 cM long, with a mean marker density of 6.5 cM. The consensus map obtained by mapping 396 markers had a length of 1476.4 cM and a 3.82-cM mean marker density. We also identified the positions of 57 SSR loci previously unmapped in apple. As a result, once additional markers have been incorporated into the maps to increase saturation, the evaluation of phenotypic traits in the mapping population should facilitate linkage between molecular markers and fruit quality traits. Additionally, the constructed apple reference genetic map, with 207 SSR markers, should also assist the construction of new genetic maps and aid integration of various apple linkage maps. [ABSTRACT FROM AUTHOR]

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants. [ABSTRACT FROM AUTHOR]

Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid ( Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially ( P < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant ( P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly ( P < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03-WAF08) and late (WAF16-WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in 'Golden Delicious'. [ABSTRACT FROM AUTHOR]

Background: Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation. Results: The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in 'Granny Smith' and 'Royal Gala' apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms. Conclusion: The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars. [ABSTRACT FROM AUTHOR]

Diacylglycerol kinase (DGK) is a pivotal enzyme that phosphorylates diacylglycerol (DAG) to form phosphatidic acid (PA). The production of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a critical signaling process in animal and plant cells. Next to PLD, DGK is the second most important generator of PA in biotic and abiotic stress responses. We identified 8 DGK members within the apple genome and all of their putative proteins contain one DGK catalytic domain and one DGK accessory domain. Four coding sequences were confirmed by cloning from Malus prunifolia . Phylogenetic and gene structure analyses showed that the apple DGK genes could be assigned to Clusters I, II, or III. Expression analysis of 6 of them revealed that their transcript levels were highest in stems. Some apple DGK genes were also significantly up-regulated in response to salt and drought stresses. This suggested their possible roles in plant defenses against environmental challenges. As a first step toward genome-wide analyses of the DGK genes in woody plants, our results imply that apple DGK genes are involved in the signaling of stress responses. These findings will contribute to further functional dissection of this gene family. [ABSTRACT FROM AUTHOR]

Apple production depends on the fulfilment of a chilling requirement for bud dormancy release. Insufficient winter chilling results in irregular and suboptimal bud break in the spring, with negative impacts on apple yield. Trees from apple cultivars with contrasting chilling requirements for bud break were used to investigate the expression of the entire set of apple genes in response to chilling accumulation in the field and controlled conditions. Total RNA was analysed on the AryANE v.1.0 oligonucleotide microarray chip representing 57 000 apple genes. The data were tested for functional enrichment, and differential expression was confirmed by real-time PCR. The largest number of differentially expressed genes was found in samples treated with cold temperatures. Cold exposure mostly repressed expression of transcripts related to photosynthesis, and long-term cold exposure repressed flavonoid biosynthesis genes. Among the differentially expressed selected candidates, we identified genes whose annotations were related to the circadian clock, hormonal signalling, regulation of growth, and flower development. Two genes, annotated as FLOWERING LOCUS C-like and MADS AFFECTING FLOWERING, showed strong differential expression in several comparisons. One of these two genes was upregulated in most comparisons involving dormancy release, and this gene's chromosomal position co-localized with the confidence interval of a major quantitative trait locus for the timing of bud break. These results indicate that photosynthesis and auxin transport are major regulatory nodes of apple dormancy and unveil strong candidates for the control of bud dormancy. [ABSTRACT FROM AUTHOR]

Background: Primary roots (radicles) represent the first visible developmental stages of the plant and are crucial for nutrient supply and the integration of environmental signals. Few studies have analyzed primary roots at a molecular level, and were mostly limited to Arabidopsis. Here we study the primary root transcriptomes of standard type, heterozygous columnar and homozygous columnar apple (Malus x domestica) by RNA-Seq and quantitative real-time PCR. The columnar growth habit is characterized by a stunted main axis and the development of short fruit spurs instead of long lateral branches. This compact growth possesses economic potential because it allows high density planting and mechanical harvesting of the trees. Its molecular basis has been identified as a nested Gypsy-44 retrotransposon insertion; however the link between the insertion and the phenotype as well as the timing of the phenotype emergence are as yet unclear. We extend the transcriptomic studies of columnar tissues to the radicles, which are the earliest developmental stage and investigate whether homozygous columnar seedlings are viable. Results: Radicles mainly express genes associated with primary metabolism, growth and development. About 200 genes show differential regulation in a comparison of heterozygous columnar radicles with non-columnar radicles, whereas the comparison of homozygous columnar radicles with non-columnar radicles yields about 300 differentially regulated genes. Genes involved in cellulose and phenylpropanoid biosynthesis, cell wall modification, transcription and translation, ethylene and jasmonate biosynthesis are upregulated in columnar radicles. Genes in the vicinity of the columnar-specific Gypsy-44 insertion experience an especially strong differential regulation: the direct downstream neighbor, dmr6-like, is downregulated in heterozygous columnar radicles, but strongly upregulated in columnar shoot apical meristems. Conclusions: The transcriptomic profile of primary roots reflects their pivotal role in growth and development. Homozygous columnar embryos are viable and form normal radicles under natural conditions, and selection towards heterozygous plants most likely occurs due to breeders' preferences. Cell wall and phytohormone biosynthesis and metabolism experience differential regulation in columnar radicles. Presumably the first step of the differential regulation most likely happens within the region of the retrotransposon insertion and its tissue-specificity suggests involvement of one (or several) tissue-specific regulator(s). [ABSTRACT FROM AUTHOR]

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, Mβ, 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. [ABSTRACT FROM AUTHOR]

Apple ( Malus × domestica Borkh.) is the most important deciduous tree fruit crop grown around the world. Comparisons of gene expression profiles from different tissues, conditions or cultivars are valuable scientific tools to better understand the gene expression changes behind important silvicultural and nutritional traits. However, the accuracy of techniques employed to access gene expression is dependent on the evaluation of stable reference genes for data normalization to avoid statistical significance undue or incorrect conclusions. The objective of this work was to select the best genes to be used as references for gene expression studies in apple trees by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Vegetative and reproductive tissues of the apple 'Gala' cultivar were evaluated during their seasonal cycle of growth and dormancy. The expression of 23 traditional housekeeping genes or genes suggested as constitutive by microarray data was investigated. Tested combinations of primers allowed the specific amplification and the generation of suitable efficiency curves for gene expression studies by RT-qPCR. Gene stability was determined by two different statistical descriptors, geNorm and NormFinder. The known variable PAL gene expression was used to validate selected normalizers. Results obtained allowed us to conclude that MDH, SAND, THFS, TMp1 and WD40 are the best reference genes to accurately normalize the relative transcript abundances using RT-qPCR in various tissues of apple. [ABSTRACT FROM AUTHOR]

Abstract: Cystatins or phytocystatins (PhyCys) comprise a family of plant-specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes as well as defense against biotic or abiotic stresses. However, information about this family is limited in apple. We identified 26 PhyCys genes within the entire apple genome. They were clustered into three distinct groups distributed across several chromosomes. All of their putative proteins contained one or two typical cystatin domains, which shared the characteristic motifs of PhyCys. Eight selected genes displayed differential expression patterns in various tissues. Moreover, their transcript levels were also up-regulated significantly in leaves during maturation, senescence or in response to treatment with one or more abiotic stresses. Our results indicated that members of this family may function in tissue development, leaf senescence, and adaptation to adverse environments in apple. [Copyright &y& Elsevier]