Profiling of phytohormones in apple fruit and buds regarding their role as potential regulators of flower bud formation.

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

No Evidence of Unexpected Transgenic Insertions in T1190 - A Transgenic Apple Used in Rapid Cycle Breeding - Following Whole Genome Sequencing.

Rapid cycle breeding uses transgenic early flowering plants as crossbreed parents to facilitate the shortening of breeding programs for perennial crops with long-lasting juvenility. Rapid cycle breeding in apple was established using the transgenic genotype T1190 expressing the BpMADS4 gene of silver birch. In this study, the genomes of T1190 and its non-transgenic wild-type PinS (F1-offspring of 'Pinova' and 'Idared') were sequenced by Illumina short-read sequencing in two separate experiments resulting in a mean sequencing depth of 182× for T1190 and 167× for PinS. The sequencing revealed 8,450 reads, which contain sequences of ≥20 bp identical to the plant transformation vector. These reads were assembled into 125 contigs, which were examined to see whether they contained transgenic insertions or if they are not using a five-step procedure. The sequence of one contig represents the known T-DNA insertion on chromosome 4 of T1190. The sequences of the remaining contigs were either equally present in T1190 and PinS, their part with sequence identity to the vector was equally present in apple reference genomes, or they seem to result from endophytic contaminations rather than from additional transgenic insertions. Therefore, we conclude that the transgenic apple plant T1190 contains only one transgenic insertion, located on chromosome 4, and shows no further partial insertions of the transformation vector. Accession Numbers: JQ974028.1.

Corrigendum: Genes Involved in Stress Response and Especially in Phytoalexin Biosynthesis Are Upregulated in Four Malus Genotypes in Response to Apple Replant Disease.

[This corrects the article DOI: 10.3389/fpls.2019.01724.].

Toward Systematic Understanding of Flower Bud Induction in Apple: A Multi-Omics Approach.

The induction of flower buds in apple (Malus × domestica Borkh.) is tightly connected to biennial bearing, which is characterized by alternating years with high (ON) and low or no (OFF) crop loads. In order to study this irregular cropping behavior, spur buds from ON- and OFF-trees of the biennial-bearing cultivar 'Fuji' and the regular bearing cultivar 'Gala' were collected. First, the time of flower bud initiation was precisely determined for both cultivars by histological analysis. Moreover, for a systematic understanding of flower bud induction in apple, the physiological and molecular mechanisms within the bud tissue were evaluated over four weeks prior to flower bud initiation by employing a multi-omics approach, including RNA sequencing, proteomic and metabolic profiling. Gene and protein enrichment analysis detected physiological pathways promoting and inhibiting early flower bud development. Metabolic profiles from the cropping treatments revealed a greater abundance of thiamine, chlorogenic acid, and an adenine derivative in spur buds from OFF-trees, whereas tryptophan was more abundant in the buds collected from ON-trees. Cultivar comparison indicated that chlorogenic acid was more abundant in 'Gala' than in 'Fuji' spur buds, whereas the opposite effect was found for tryptophan. Genes controlling tryptophan biosynthesis were not affected by ON- and OFF-treatments, but genes assigned to the metabolism of tryptophan into indoleacetate were differentially expressed between cultivars and treatments. The multi-omics approach permitted analyzing complex plant metabolic processes involved in early flower bud development and more specifically presumably in flower bud induction by tracing some pathways from gene to product level.

Transcriptional profile of AvrRpt2EA-mediated resistance and susceptibility response to Erwinia amylovora in apple.

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor.

Genetic Diversity and Genome Size Variability in the Russian Genebank Collection of Tea Plant [Camellia sinensis (L). O. Kuntze].

The tea collection of the FRC SSC RAS (Sochi, Maykop in Russia) represents one of the northernmost germplasm comprising a number of locally derived cultivars and ɣ-irradiation mutants. The latter are often characterized by larger genome size, which may lead to better adaptation to biotic and abiotic stress. Such genotypes may be a valuable genetic resource for better adaptability to extreme environmental conditions, which could enable tea cultivation outside global growing regions. Microsatellite markers are often the best choice for genetic diversity analysis in genebank collections. However, their use in polyploid species is questionable because simple sequence repeat (SSR) allele dosage cannot be readily determined. Therefore, the efficiency of SSR and start codon targeted (SCoT) markers was investigated using 43 selected cultivars from the Russian genebank collection derived from mutant breeding and clonal selection. Previously, the increase in genome size was confirmed in 18 mutants within this collection. Despite the presence of polyploid tea genotypes, our study revealed higher efficiency of SSR markers than SCoT markers. Subsequent SSR analysis of the 106 genotypes in the Russian genebank collection revealed three distinct genetic clusters after STRUCTURE analysis. Greater genetic variation was observed within genetic clusters than between clusters, indicating low genetic variation between collections. Nevertheless, the northernmost tea collection exhibited a greater genetic distance from the other two clusters than they did from each other. Close genetic relationships were found between many cultivars with particularly large leaves and mutant forms. Pearson's correlation analysis revealed a significant, moderate correlation between genome size and leaf area size. Our study shows that microsatellite fingerprinting is useful to estimate the genetic diversity and genetic background of tea germplasm in Russia despite polyploid tea accessions. Thus, the results of our study contribute to the development of future tea germplasm conservation strategies and modern tea breeding programs.

Physiological, biochemical and genetic responses of Caucasian tea (Camellia sinensis (L.) Kuntze) genotypes under cold and frost stress.

BACKGROUND: Cold and frost are two serious factors limiting the yield of many crops worldwide, including the tea plant (Camellia sinensis (L.) Kuntze). The acclimatization of tea plant from tropical to temperate climate regions resulted in unique germplasm in the North-Western Caucasus with extremely frost-tolerant genotypes. METHODS: The aim of the current research was to evaluate the physiological, biochemical and genetic responses of tolerant and sensitive tea cultivars exposed to cold (0 to +2 °C for 7 days) and frost (-6 to -8 °C for 5 days). Relative water content, cell membranes integrity, pH of the cell sap, water soluble protein, cations, sugars, amino acids were measured under cold and frost. Comparative expression of the following genes ICE1, CBF1, WRKY2, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, SnRK1.1, SnRK1.2, SnRK1.3, bHLH7, bHLH43, P5CS, LOX1, LOX6, LOX7 were analyzed. RESULTS: We found elevated protein (by 3-4 times) and cations (potassium, calcium and magnesium) contents in the leaves of both cultivars under cold and frost treatments. Meanwhile, Leu, Met, Val, Thr, Ser were increased under cold and frost, however tolerant cv. Gruzinskii7 showed earlier accumulation of these amino acids. Out of 18 studied genes, 11 were expressed at greater level in the frost- tolerant cultivar comparing with frost-sensitive one: ICE1, CBF1, WRKY2, DHN2, NAC17, NAC26, SnRK1.1, SnRK1.3, bHLH43, P5CS and LOX6. Positive correlations between certain amino acids namely, Met, Thr, Leu and Ser and studied genes were found. Taken together, the revealed cold responses in Caucasian tea cultivars help better understanding of tea tolerance to low temperature stress and role of revealed metabolites need to be further evaluated in different tea genotypes.

The MADS-Box Gene MdDAM1 Controls Growth Cessation and Bud Dormancy in Apple.

Apple trees require a long exposure to chilling temperature during winter to acquire competency to flower and grow in the following spring. Climate change or adverse meteorological conditions can impair release of dormancy and delay bud break, hence jeopardizing fruit production and causing substantial economic losses. In order to characterize the molecular mechanisms controlling bud dormancy in apple we focused our work on the MADS-box transcription factor gene MdDAM1. We show that MdDAM1 silencing is required for the release of dormancy and bud break in spring. MdDAM1 transcript levels are drastically reduced in the low-chill varieties 'Anna' and 'Dorsett Golden' compared to 'Golden Delicious' corroborating its role as a key genetic factor controlling the release of bud dormancy in Malus species. The functional characterization of MdDAM1 using RNA silencing resulted in trees unable to cease growth in winter and that displayed an evergrowing, or evergreen, phenotype several years after transgenesis. These trees lost their capacity to enter in dormancy and produced leaves and shoots regardless of the season. A transcriptome study revealed that apple evergrowing lines are a genocopy of 'Golden Delicious' trees at the onset of the bud break with the significant gene repression of the related MADS-box gene MdDAM4 as a major feature. We provide the first functional evidence that MADS-box transcriptional factors are key regulators of bud dormancy in pome fruit trees and demonstrate that their silencing results in a defect of growth cessation in autumn. Our findings will help producing low-chill apple variants from the elite commercial cultivars that will withstand climate change.

Mapping of fire blight resistance in Malus ×robusta 5 flowers following artificial inoculation.

BACKGROUND: Although the most common path of infection for fire blight, a severe bacterial disease on apple, is via host plant flowers, quantitative trait loci (QTLs) for fire blight resistance to date have exclusively been mapped following shoot inoculation. It is not known whether the same mechanism underlies flower and shoot resistance. RESULTS: We report the detection of a fire blight resistance QTL following independent artificial inoculation of flowers and shoots on two F1 segregating populations derived from crossing resistant Malus ×robusta 5 (Mr5) with susceptible 'Idared' and 'Royal Gala' in experimental orchards in Germany and New Zealand, respectively. QTL mapping of phenotypic datasets from artificial flower inoculation of the 'Idared' × Mr5 population with Erwinia amylovora over several years, and of the 'Royal Gala' × Mr5 population in a single year, revealed a single major QTL controlling floral fire blight resistance on linkage group 3 (LG3) of Mr5. This QTL corresponds to the QTL on LG3 reported previously for the 'Idared' × Mr5 and an 'M9' × Mr5 population following shoot inoculation in the glasshouse. Interval mapping of phenotypic data from shoot inoculations of subsets from both flower resistance populations re-confirmed that the resistance QTL is in the same position on LG3 of Mr5 as that for flower inoculation. These results provide strong evidence that fire blight resistance in Mr5 is controlled by a major QTL on LG3, independently of the mode of infection, rootstock and environment. CONCLUSIONS: This study demonstrates for the first time that resistance to fire blight caused by Erwinia amylovora is independent of the mode of inoculation at least in Malus ×robusta 5.

High crop load and low temperature delay the onset of bud initiation in apple.

The reproductive cycle of apple (Malus × domestica Borkh.) starts with the induction of floral development, however, first morphological changes within the bud appear during the following period of bud initiation. This study identifies the onset and duration of bud initiation in the apple cultivars 'Fuji' and 'Gala', characterized by biennial and non-biennial bearing behaviour, respectively, and describes the effect of crop load and heat accumulation on the temporal pattern of floral development. The onset of flower bud initiation in heavy cropping 'Gala' trees was delayed for 20 days compared to trees with no crop load, but the rate of initiation was not affected by crop load. Bud initiation on heavy cropping 'Fuji' trees was minor, whereas trees with no crop load started initiating buds 19 days earlier than those of 'Gala' despite the same cropping status and growing degree hours in a given year. The onset of bud initiation in 'Fuji' 'off' trees was 5 and 20 days after summer solstice, respectively, in two consecutive growing seasons, suggesting that this process is driven by heat accumulation rather than by daylength. The results indicate, that the genetic make-up of the cultivar determines the onset of bud initiation. This can be delayed by increasing crop loads and low temperatures at the beginning of the flower formation process.

Genes Involved in Stress Response and Especially in Phytoalexin Biosynthesis Are Upregulated in Four Malus Genotypes in Response to Apple Replant Disease.

Apple replant disease (ARD) is a soil-borne disease, which is of particular importance for fruit tree nurseries and fruit growers. The disease manifests by a poor vegetative development, stunted growth, and reduced yield in terms of quantity and quality, if apple plants (usually rootstocks) are replanted several times at the same site. Genotype-specific differences in the reaction of apple plants to ARD are documented, but less is known about the genetic mechanisms behind this symptomatology. Recent transcriptome analyses resulted in a number of candidate genes possibly involved in the plant response. In the present study, the expression of 108 selected candidate genes was investigated in root and leaf tissue of four different apple genotypes grown in untreated ARD soil and ARD soil disinfected by γ-irradiation originating from two different sites in Germany. Thirty-nine out of the 108 candidate genes were differentially expressed in roots by taking a p-value of < 0.05 and a fold change of > 1.5 as cutoff. Sixteen genes were more than 4.5-fold upregulated in roots of plants grown in ARD soil. The four genes MNL2 (putative mannosidase); ALF5 (multi antimicrobial extrusion protein); UGT73B4 (uridine diphosphate (UDP)-glycosyltransferase 73B4), and ECHI (chitin-binding) were significantly upregulated in roots. These genes seem to be related to the host plant response to ARD, although they have never been described in this context before. Six of the highly upregulated genes belong to the phytoalexin biosynthesis pathway. Their genotype-specific gene expression pattern was consistent with the phytoalexin content measured in roots. The biphenyl synthase (BIS) genes were found to be useful as early biomarkers for ARD, because their expression pattern correlated well with the phenotypic reaction of the Malus genotypes investigated.

Towards map-based cloning of FB_Mfu10: identification of a receptor-like kinase candidate gene underlying the Malus fusca fire blight resistance locus on linkage group 10.

Breeding for resistance against the destructive fire blight disease of apples is the most sustainable strategy to control the menace of this disease, and has become increasingly important in European apple breeding programs. Since most cultivars are susceptible, wild accessions have been explored for resistance with quantitative trait loci detected in a few wild species. Fire blight resistance of Malus fusca was described following phenotypic evaluations with a C-type strain of Erwinia amylovora, Ea222_JKI, and the detection of a major QTL on chromosome 10 (Mfu10) of this crabapple. The stability of the resistance of M. fusca and Mfu10 has been evaluated using two other strains, the highly aggressive Canadian S-type strain-Ea3049, and the avrRpt2EA mutant-ZYRKD3-1, both of which overcome the resistance of Malus ×robusta 5, a wild species accession with an already described fire blight resistance gene. To pave the way for positional cloning of the underlying fire blight resistance gene of M. fusca, we have fine mapped the QTL region on linkage group 10 using 1888 individuals and 23 newly developed molecular markers, thus delimiting the interval of interest to 0.33 cM between markers FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22. Tightly linked SSR markers are suitable for marker-assisted selection in breeding programs. Furthermore, a bacterial artificial chromosome (BAC) clone spanning FB_Mfu10 region was isolated and sequenced. One putative fire blight resistance candidate gene of M. fusca was predicted on the sequence of BAC 46H22 within the resistance region that encodes B-lectin and serine/threonine kinase domains.

A Single Effector Protein, AvrRpt2EA, from Erwinia amylovora Can Cause Fire Blight Disease Symptoms and Induces a Salicylic Acid-Dependent Defense Response.

The AvrRpt2EA effector protein of Erwinia amylovora is important for pathogen recognition in the fire blight-resistant crabapple Malus × robusta 5; however, little is known about its role in susceptible apples. To study its function in planta, we expressed a plant-optimized version of AvrRpt2EA driven by a heat shock-inducible promoter in transgenic plants of the fire blight-susceptible cultivar Pinova. After induced expression of AvrRpt2EA, transgenic lines showed shoot necrosis and browning of older leaves, with symptoms similar to natural fire blight infections. Transgenic expression of this effector protein resulted in an increase in the expression of the salicylic acid (SA)-responsive PR-1 gene but, also, in the levels of SA and its derivatives, with diverse kinetics in leaves of different ages. In contrast, no increase of expression levels of VSP2 paralogs, used as marker genes for the activation of the jasmonic acid (JA)-dependent defense pathway, could be detected, which is in agreement with metabolic profiling of JA and its derivatives. Our work demonstrates that AvrRpt2EA acts as a virulence factor and induces the formation of SA and SA-dependent systemic acquired resistance.

Generation of advanced fire blight-resistant apple (Malus × domestica) selections of the fifth generation within 7 years of applying the early flowering approach.

MAIN CONCLUSION: The approach presented here can be applied to reduce the time needed to introduce traits from wild apples into null segregant advanced selections by one-fourth. Interesting traits like resistances to pathogens are often found within the wild apple gene pool. However, the long juvenile phase of apple seedlings hampers the rapid introduction of these traits into new cultivars. The rapid crop cycle breeding approach used in this paper is based on the overexpression of the birch (Betula pendula) MADS4 transcription factor in apple. Using the early flowering line T1190 and 'Evereste' as source of the fire blight resistance (Fb_E locus), we successfully established 18 advanced selections of the fifth generation in the greenhouse within 7 years. Fifteen individuals showed the habitus expected of a regular apple seedling, while three showed very short internodes. The null segregants possessing a regular habitus maintained the high level of fire blight resistance typical for 'Evereste'. Using SSR markers, we estimated the percentage of genetic drag from 'Evereste' still associated with Fb_E on linkage group 12 (LG12). Eight out of the 18 selections had only 4% of 'Evereste' genome left. Since genotypes carrying the apple scab resistance gene Rvi6 and the fire blight resistance QTL Fb_F7 were used as parents in the course of the experiments, these resistances were also identified in some of the null segregants. One seedling is particularly interesting as, beside Fb_E, it also carries Fb_F7 heterozygously and Rvi6 homozygously. If null segregants obtained using this method will be considered as not genetically modified in Europe, as is already the case in the USA, this genotype could be a very promising parent for breeding new fire blight and scab-resistant apple cultivars in European apple breeding programs.

Evaluation of a MdMYB10/GFP43 fusion gene for its suitability to act as reporter gene in promoter studies in Fragaria vesca L. 'Rügen'.

A Malus domestica MdMYB10 transcription factor gene was previously used as visible marker for successful plant transformation. We combined the MdMYB10 transcription factor gene with a GFP gene to test its viability as a non-destructive, visual, double reporter system for functional promoter studies in transgenic strawberry plants. The GFP gene was fused to MdMYB10 to provide evidence for promoter activity in red colored cells of transformed plant tissue and to exclude artefacts resulting from stress response or due to other environmental cues. To test this system in a first approach, we evaluated the MdMYB10-GFP43 construct in transgenic strawberries in combination with two constitutive promoters of varying strength, the strong CaMV 35S promoter and a weak flavonoid 3'-hydroxylase (F3'H) promoter isolated from the ornamental plant Cosmos sulphureus. Agrobacterium tumefaciens mediated transformation of Fragaria vesca with the MdMYB10-GFP43 construct combined with the CaMV 35S or F3'H promoter sequences resulted in the regeneration of 6 and 4 transgenic lines, respectively. A complete red coloration of all plant organs was found in four out of six transgenic lines harboring the 35S-MdMYB10-GFP43 construct. Less red coloration of plant organs was found for lines transformed with the F3'H-MdMYB10-GFP43 construct. The MdMYB10 gene shows only limited suitability as a reporter gene for promoter studies in strawberries because weak promoter activity is difficult to distinguish, particularly in tissues showing a strongly colored background such as green leaves. GFP specific fluorescence signals were detectable neither in tissue strongly expressing MdMYB10 nor in green tissue of any transgenic line. The reason for this remained unclear but it can be excluded that it was due to incorrect splicing.

Spatial and Temporal Localization of Flavonoid Metabolites in Strawberry Fruit (Fragaria × ananassa).

Flavonoids are important metabolites in strawberries (Fragaria × ananassa) because they accomplish an extensive collection of physiological functions and are valuable for human health. However, their localization within the fruit tissue has not been extensively explored. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to shed light on the spatial distribution of flavonoids during fruit development. One wild-type (WT) and two transgenic lines were compared, wherein the transgenic enzymes anthocyanidin reductase (ANRi) and flavonol synthase (FLSi), respectively, were down-regulated using an RNAi-based silencing approach. In most cases, fruit development led to a reduction of the investigated flavonoids in the fruit tissue; as a consequence, they were exclusively present in the skin of mature red fruits. In the case of (epi)catechin dimer, both the ANRi and the WT phenotypes revealed low levels in mature red fruits, whereas the ANRi line bore the lowest relative concentration, as analyzed by liquid chromatography-electrospray ionization multiple-step mass spectrometry (LC-ESI-MSn).

TERMINAL FLOWER1 is a breeding target for a novel everbearing trait and tailored flowering responses in cultivated strawberry (Fragaria × ananassa Duch.).

The effects of daylength and temperature on flowering of the cultivated octoploid strawberry (Fragaria × ananassa Duch.) have been studied extensively at the physiological level, but information on the molecular pathways controlling flowering in the species is scarce. The flowering pathway has been studied at the molecular level in the diploid short-day woodland strawberry (F. vesca L.), in which the FLOWERING LOCUS T1 (FvFT1)-SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (FvSOC1)-TERMINAL FLOWER1 (FvTFL1) pathway is essential for the correct timing of flowering. In this work, we show by transgenic approach that the silencing of the floral repressor FaTFL1 in the octoploid short-day cultivar 'Elsanta' is sufficient to induce perpetual flowering under long days without direct changes in vegetative reproduction. We also demonstrate that although the genes FaFT1 and FaSOC1 show similar expression patterns in different cultivars, the regulation of FaTFL1 varies widely from cultivar to cultivar and is correlated with floral induction, indicating that the transcription of FaTFL1 occurs at least partially independently of the FaFT1-FaSOC1 module. Our results indicate that changing the expression patterns of FaTFL1 through biotechnological or conventional breeding approaches could result in strawberries with specific flowering and runnering characteristics including new types of everbearing cultivars.

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

MAIN CONCLUSION: Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3'-hydroxylase (F3'H). Using recombinant F3'H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3'H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.

Integration of BpMADS4 on various linkage groups improves the utilization of the rapid cycle breeding system in apple.

Rapid cycle breeding in apple is a new approach for the rapid introgression of agronomically relevant traits (e.g. disease resistances) from wild apple species into domestic apple cultivars (Malus × domestica Borkh.). This technique drastically shortens the long-lasting juvenile phase of apple. The utilization of early-flowering apple lines overexpressing the BpMADS4 gene of the European silver birch (Betula pendula Roth.) in hybridization resulted in one breeding cycle per year. Aiming for the selection of non-transgenic null segregants at the end of the breeding process, the flower-inducing transgene and the gene of interest (e.g. resistance gene) that will be introgressed by hybridization need to be located on different chromosomes. To improve the flexibility of the existing approach in apple, this study was focused on the development and characterization of eleven additional BpMADS4 overexpressing lines of four different apple cultivars. In nine lines, the flowering gene was mapped to different linkage groups. The differences in introgressed T-DNA sequences and plant genome deletions post-transformation highlighted the unique molecular character of each line. However, transgenic lines demonstrated no significant differences in flower organ development and pollen functionality compared with non-transgenic plants. Hybridization studies using pollen from the fire blight-resistant wild species accession Malus fusca MAL0045 and the apple scab-resistant cultivar 'Regia' indicated that BpMADS4 introgression had no significant effect on the breeding value of each transgenic line.

Phenotypic and genetic analysis of the German Malus Germplasm Collection in terms of type 1 and type 2 red-fleshed apples.

Red fruit flesh is a desirable trait in apple breeding, because red-fleshed apples are a novelty and therefore considered to be more attractive to consumers and contain more health beneficial compounds. The red fruit flesh coloration is based on an increased level of cyanidin 3-galactoside, an anthocyanin whose biosynthesis is regulated by the MYB-type transcription factors MdMYB10 or MdMYB110a, respectively. A repeated segment in the MdMYB10 promoter allele R6 results in a gain-of-function mutation visible as red pigmentation of fruit skin and flesh and all vegetative tissues. Red-fleshed apple genotypes containing this R6 allele belong to the type 1 red-fleshed apple, which is known to be linked to some negative traits like astringent taste and internal flesh browning disorder. In type 2 red-fleshed apples the fruit flesh coloration is not inevitably linked with skin and leaf color. This red-fleshed apple phenotype, which is a result of increased expression of MdMYB110a, seems to be more useful for breeding, but it can be found rather seldom. In the present study 357 Malus accessions of the German Malus Germplasm Collection were evaluated for red fruit flesh coloration and the presence of the MdMYB10 R1 (not mutated) and R6 promoter alleles. Among them a total of 40 accessions were identified which contain the R6 allele. 37 accessions showed a red coloration of the fruit flesh. All these accessions belong to type 1 red-fleshed apple. No type 2 red-fleshed apple could be found. Three accessions with R6 allele had non-red-fleshed apples. 312 other non-red-fleshed accessions contained only the R1 allele. Five non-red-fleshed accessions contained a new promoter allele with an unexpected size of ~1 kbp. Sequencing of this allele detected the insertion of a non-autonomous apple transposon.