Identification of self-incompatibility-related glycoproteins in styles of apple (Malus x domestica)

In this study, stylar proteins of apple (Malus x domestica) which correlate with known intervarietal incompatibility relationships and have similar characteristics to the S-glycoproteins of Japanese pear (Pyrus serotina) were surveyed by two-dimensional gel electrophoresis (2D-PAGE). Varietal differences were detected in a group of glycoproteins having Mrs and pIs similar to those of the S-glycoproteins of Japanese pear. 2D-PAGE profiles of these glycoproteins were correlated with intervarietal incompatibility relationships. These glycoproteins reacted with antiserum raised against the S ⁴-glycoprotein of Japanese pear, a result suggesting that they may be the products of S-alleles in styles of apple. On the basis of the profiles of the putative S-glycoproteins, S-genotypes were proposed for each of the apple cultivars examined.     

Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis

Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO, and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.     

Architecture and size relations: an essay on the apple (Malus x domestica, Rosaceae) tree

The influence of tree size independent of age on some architectural features (annual shoot length, lateral branching, flowering) was investigated on 4-yr-old apple (Malus × domestica) trees either own-rooted or grafted on the dwarfing rootstock M.9, giving rise to large and small trees, respectively. Tree size significantly affected the length of the first annual shoot of bottom branches with a lesser effect on the subsequent annual shoots of the same branches and on branches situated higher in the tree canopy. The linear regression parameters, i.e., slopes and intercepts, between annual shoot length and number of growing laterals were affected by the genotype and, depending on genotype, by tree size. Flowering was generally lower, delayed, and more irregular on large trees compared to small trees, with on average similar ranking of genotypes regardless of tree size. This study provides evidence for a specific effect of tree size, as affected by the root system, on architectural development of the apple tree regardless of the genotype. From an architectural viewpoint, the dwarfing mechanism could be interpreted as a faster physiological aging essentially related to the reduction in length of the first annual shoot of bottom branches and the high flowering on this shoot.     

Ribonuclease inhibitors in Malus x domestica (common apple): isolation and partial characterization

A ribonuclease inhibitory activity was detected in the fruits of common apple, Malus x domestica, cv. Fuji, and purified by affinity chromatography on ribonuclease A-Sepharose. It inhibited hydrolysis of cyclic-2':3'-CMP by bovine pancreatic ribonuclease A with an apparent inhibition constant of about 5 x 10(-8) M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein gave two peaks corresponding to the mass numbers of 55,658 and 62,839, while three bands of 43-, 34-, and 21-kDa were detected by SDS-PAGE. These results suggested that the inhibitor preparation was a mixture of two proteins comprised of 43- and 21-kDa subunits or of 34- and 21-kDa subunits. Attempts to separate these two proteins were unsuccessful. Amino acid composition and N-terminal amino acid sequence of these subunits were also identified and N-terminal sequences showed some similarity to that of cottonseed storage globulin. The significance of the presence of ribonuclease inhibitors in apple fruits is not clear, but it might allow some speculation about their possible involvement in the control of the self-incompatibility ribonuclease of Rosaceae plants.     

Characterisation of the DELLA subfamily in apple (Malus x domestica Borkh.)

The hormone gibberellic acid (GA) regulates growth and development throughout the plant life cycle. DELLA proteins are key components of the GA signalling pathway and act to repress GA responses. The “DELLA” amino acid motif is highly conserved among diverse species and is essential for GA-induced destruction of DELLA proteins, which relieves repression. Six genes encoding the DELLA motif were identified within an apple expressed sequence tag (EST) database. Full-length cDNA clones were obtained by RACE and these were designated MdRGL1a/b, MdRGL2a/b, and MdRGL3a/b. Sequence alignment of the predicted proteins indicates that the MdDELLAs are 37–93% homologous to one another and 44–65% to the Arabidopsis DELLAs. The MdDELLAs cluster into three pairs, which reflect the presumed allopolyploid origins of the Maloideae. Expression analysis using quantitative real-time PCR indicates that all three pairs of MdDELLA mRNAs are expressed at the highest levels in summer arrested shoot tips and in autumn vegetative buds. Transgenic Arabidopsis expressing MdRGL2a have smaller leaves and shorter stems, take longer to flower in short days, and exhibit a reduced response to exogenous GA₃, indicating significant conservation of gene function between DELLA proteins from apple and Arabidopsis. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Microsatellite markers spanning the apple (Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database () to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.E. Silfverberg-Dilworth and C. L. Matasci contributed equally to this work.     

Development and characterisation of 140 new microsatellites in apple (Malus x domestica Borkh.)

The availability of suitable genetic markers is essential to efficiently select and breed apple varieties of high quality and with multiple disease resistances. Microsatellites (simple sequence repeats, SSR) are very useful in this respect since they are codominant, highly polymorphic, abundant and reliably reproducible. Over 140 new SSR markers have been developed in apple and tested on a panel of 7 cultivars and 1 breeding selection. Their high level of polymorphism is expressed with an average of 6.1 alleles per locus and an average heterozygosity (H) of 0.74. Of all SSR markers, 115 have been positioned on a genetic linkage map of the cross Fiesta × Discovery. As a result, all 17 linkage groups, corresponding to the 17 chromosomes of apple, were identified. Each chromosome carries at least two SSR markers, allowing the alignment of any apple molecular marker map both with regard to identification as well as to orientation of the linkage groups. To test the degree of conservation of the SSR flanking regions and the transferability of the SSR markers to other Rosaceae species, 15 primer pairs were tested on a series of Maloideae and Amygdaloideae species. The usefulness of the newly developed microsatellites in genetic mapping is demonstrated by means of the genetic linkage map. The possibility of constructing a global apple linkage map and the impact of such a number of microsatellite markers on gene and QTL mapping is discussed.     

Inheritance of pollen enzymes and polyploid origin of apple (Malus x domestica Borkh.)

Summary Electrophoresis of 7 pollen enzymes was applied to 5 progenies from controlled crosses and one self-progeny of apple. Segregation data were examined according to three kinds of hypotheses: monogenic disomic, bigenic disomic and tetrasomic inheritance Twenty codominant alleles and a recessive null were identified. Results provided evidence of bigenic disomic inheritance in most cases: 6 pairs of homoeologous loci carrying identical homoeoalleles were revealed; only 2 enzymes exhibited a simple monogenic control. Preferential pairing between pairs of homologous chromosomes in meiosis can be postulated. These results indicated an allopolyploid origin of apple genome. Fixed heterozygosity occurred for several enzymes, which is a typical feature of allopolyploidy. Loss of duplicate gene expression can account for the monogenic control of 2 of the enzymes.The results reported in this paper are part of a thesis by the first author for the degree of Docteur Ingénieur     

Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of reference genes relied on using homology to reference genes in model species. In this study, a genomics approach was used to identify apple genes with low variance in expression in 217 messenger RNA (mRNA)-seq data sets covering different tissues, during fruit development, and treated with a range of different stress conditions. Ten potential reference genes were chosen for validation by quantitative PCR (qPCR) over 29 different tissue types and treatments. From the combined mRNA-seq and qPCR results, three potential reference genes are proposed that can be used as good controls for PCR based expression studies. The three genes show homology to lipid transfer proteins, phytochrome protein phosphatase and the ubiquitination pathway. With the progression of research away from non-model species, this approach provides a robust method for selecting candidate genes for use as reference genes in qPCR.     

Potential polyphenol markers of phase change in apple (Malus domestica)

In order to identify potential biochemical markers that can be used as indicators for phase change, the dynamics of polyphenolic compounds across apple seedlings (Malus domestica, Jonathan x Golden Delicious) were analyzed in this study by high performance liquid chromatography. Precocious flowering was induced by foliar sprays of plant growth regulators. Qualitative changes in the concentration of polyphenols were observed at node nos. 50, 80 and 120. Spontaneous and induced flowering was found at node nos. 122 and 77. It was reasonable to conclude that node no. 77 represented the point of transition between the juvenile phase and the adult vegetative phase, which was marked by the presence of phloridzin in the buds. The disappearance of myricitrin in the bark and the absence of caffeic acid in the aboveground tissues were qualitative markers of the reproductive phase, which was reached at node no. 122.     

A morphological and quantitative characterization of early floral development in apple (Malus x domestica Borkh.)

Apple is an important crop and a focus of research worldwide. However, some aspects of floral commitment and morphogenesis remain unclear. A detailed characterization of bourse shoot apex development was undertaken to provide a framework for future genetic, molecular and physiological studies. Eight morphologically distinct stages of shoot apex development, prior to winter dormancy, were defined. Based on measurements of meristem diameter, two stages of vegetative development were recognized. Vegetative meristems were flat, and either narrow (stage 0) or broad (stage 1). Pronounced doming of the apex marked stage 2. During stage 3, the domed meristem initiated four to six lateral floral meristems and subtending bracts before converting to a terminal floral meristem (stage 4). The terminal floral meristem proceeded directly with bractlet and sepal initiation, while lateral floral meristems initiated bractlets (stage 5). Sepal initiation began on the basal lateral flower (stage 6) and continued in an acropetal direction until all floral meristems had completed sepal initiation (stage 7). In this study, only stage 0 and stage 7 apices were observed in dormant buds, indicating that stages 1-6 are transient. The results suggest that broadening of the apex (stage 1) is the first morphological sign of commitment to flowering.     

Induction of polyphenol gene expression in apple (Malus x domestica) after the application of a dioxygenase inhibitor

A comprehensive study of the complex polyphenol biosynthesis in developing leaves of apple ( Malus domestica ) was performed comprising gene expression, enzyme activities and polyphenol composition. During leaf development, an early increase in gene expression was observed for phenylalanine ammonia lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 2.3.1.74), flavanone 3-hydroxylase (FHT, EC 1.14.11.9) and dihydroflavonol 4-reductase/flavanone 4-reductase (DFR/FNR, EC 1.1.1.219). Their enzyme activities showed a corresponding trend during the time course. A parallel set of experiments was carried out with leaves treated with prohexadione-Ca (ProCa), which is an enzyme inhibitor of 2-oxoglutarate dependent dioxygenases (2-ODDs). ProCa is known to induce changes in polyphenol biosynthesis, which are accompanied by a reduced incidence of fire blight and scab, the two major pome fruit diseases. The application of ProCa led to an increase in activities of PAL, CHS, FHT and DFR/FNR, which was based on an enhanced gene expression. In contrast, an inhibition of gene expression was detected for anthocyanidin synthase (EC 1.14.11.19). These effects are interpreted as a feedback regulation by changed polyphenol levels. Because of the inhibition of the 2-ODDs FHT and flavonol synthase (EC 1.14.11.23), some pronounced changes in polyphenol composition were observed. Eriodictyol, the substrate of FHT, accumulated as eriodictyol-7- O -glucoside and 6"- O - trans - p -coumaroyleriodictyol 3'- O -glucoside. In addition, the 3-deoxycatechin luteoliflavan was formed which is not present in untreated apple leaves. Hence, beyond the redirection of polyphenol biosynthesis by the enzyme inhibitor, changed polyphenol levels obviously cause a distinct induction of gene expression by feedback regulation.     

Down-regulation of POLYGALACTURONASE 1 alters firmness, tensile strength and water loss in apple (Malus x domestica) fruit

ABSTRACT: BACKGROUND: While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently no direct evidence of its function. This study examined the effect of down regulation of PG1 expression in 'Royal Gala' apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. RESULTS: PG1-suppressed 'Royal Gala' apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppression also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. CONCLUSIONS: These findings confirm PG1's role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies in shown in strawberry but not in tomato. In apple PG1 also appears influence other fruit texture characters such as juiciness and water loss.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Effect of rootstock on apple (Malus domestica) tree water relations

The effects of rootstock on mid-season water relations, under orchard conditions of non-limiting soil moisture, were determined for bearing 'Empire' apple trees ( Malus domestica Borkh.) on the clonal rootstocks M9, M26, M7, MM106, and MM104 (most to least dwarfing) in their sixth and seventh growing seasons. Stem water potentials (ψ^stem) of trees on M9 and M26 were more negative at midday, under warm, sunny conditions, than were the trees on the other three rootstocks. However, change in ψ^stem per change in stem distance through the canopy (water potential gradient) did not vary among rootstocks at midday. There was no rootstock effect on diurnal variation in transpiration or stomatal conductance. Differences in water storage capacitance, relative to tree size, were determined in a separate study but did not account for the differences observed in ψ^stem. Calculated hydraulic conductivities of xylem water transport suggest that rootstocks differ in their ability to conduct water to the scion, but hydraulic conductivity of the scion was not affected by rootstock. Root-stock differences in hydraulic conductivity were not accounted for by differences in tree size.