Constitutive expression of apple endo-POLYGALACTURONASE1 in fruit induces early maturation, alters skin structure and accelerates softening.

During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening-related endo-POLYGALACTURONASE1 gene. MdPG1-overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white-skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel-associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post-harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. ß-galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development.

A MADS-box gene-induced early flowering pear (Pyrus communis L.) for accelerated pear breeding.

There have been a considerable number of studies that have successfully sped up the flowering cycle in woody perennial horticultural species. One particularly successful study in apple (Malus domestica) accelerated flowering using a silver birch (Betula pendula) APETALA1/FRUITFULL MADS-box gene BpMADS4, which yielded a good balance of vegetative growth to support subsequent flower and fruit development. In this study, BpMADS4 was constitutively expressed in European pear (Pyrus communis) to establish whether this could be used as a tool in a rapid pear breeding program. Transformed pear lines flowered within 6-18 months after grafting onto a quince (Cydonia oblonga) rootstock. Unlike the spindly habit of early flowering apples, the early flowering pear lines displayed a normal tree-like habit. Like apple, the flower appearance was normal, and the flowers were fertile, producing fruit and seed upon pollination. Seed from these transformed lines were germinated and 50% of the progeny flowered within 3 months of sowing, demonstrating a use for these in a fast breeding program.

Overexpression of PSY1 increases fruit skin and flesh carotenoid content and reveals associated transcription factors in apple (Malus × domestica).

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion  treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis.

Elevating fruit carotenoid content in apple (Malus x domestica Borkh).

Carotenoid compounds accumulate to confer coloration to plant tissues and have some established health benefits in humans. These pigments have antioxidant properties and are precursors of vitamin A, which is important for human vision. Apple is widely consumed globally, but most commercial apple cultivars have low fruit carotenoid content because these pigments accumulate mostly in the fruit skin rather than the flesh (the majority of the edible portion). Although carotenoids accumulate in the early stages of fruit development, much of this carotenoid is lost by fruit maturity as a result of low biosynthetic rate, rapid turnover of compounds and/or lack of storage capacity in these tissues. Improving apple fruit carotenoid content through traditional breeding or genetic technologies, will take a long time because of the extended juvenile phase of the trees and limited germplasm diversity within many commercial breeding programs. This process, however, can be accelerated by fundamental understanding of the apple carotenoid biosynthetic pathway and the mechanisms controlling the metabolic steps. The availability of a well annotated apple genome sequence has led to the identification of apple carotenoid gene families and potential transcription factors. This is an important step since the knowledge could be used to elevate carotenoid content either through breeding or genetic transformation techniques. Here, we provide an overview of carotenogenesis in apple and outline the methods employed to improve the carotenoid content of this horticultural crop.

microRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica).

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

Significant improvement of apple (Malus domestica Borkh.) transgenic plant production by pre-transformation with a Baby boom transcription factor.

BABY BOOM (BBM) is a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family and its expression has been shown to improve herbaceous plant transformation and regeneration. However, this improvement has not been shown clearly for tree species. This study demonstrated that the efficiency of transgenic apple (Malus domestica Borkh.) plant production was dramatically increased by ectopic expression of the MdBBM1 gene. "Royal Gala" apple plants were first transformed with a CaMV35S-MdBBM1 construct (MBM) under kanamycin selection. These MBM transgenic plants exhibited enhanced shoot regeneration from leaf explants on tissue culture media, with most plants displaying a close-to-normal phenotype compared with CaMV35S-GUS transgenic plants when grown under greenhouse conditions, the exception being that some plants had slightly curly leaves. Thin leaf sections revealed the MBM plants produced more cells than the GUS plants, indicating that ectopic-expression of MdBBM1 enhanced cell division. Transcriptome analysis showed that mRNA levels for cell division activators and repressors linked to hormone (auxin, cytokinin and brassinosteroid) signalling pathways were enhanced and reduced, respectively, in the MBM plants compared with the GUS plants. Plants of eight independent MBM lines were compared with the GUS plants by re-transforming them with an herbicide-resistant gene construct. The number of transgenic plants produced per 100 leaf explants was 0-3% for the GUS plants, 3-8% for five MBM lines, and 20-30% for three MBM lines. Our results provided a solution for overcoming the barriers to transgenic plant production in apple, and possibly in other trees.

RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees.

DORMANCY-ASSOCIATED MADS-box (DAM) and SHORT VEGETATIVE PHASE (SVP) genes have been implicated in the regulation of winter dormancy in perennials. Ectopic expression of apple (Malus × domestica Borkh. 'Royal Gala') DAM and SVP genes delays budbreak and constrains lateral shoot outgrowth. In this study, we used RNA interference (RNAi) to simultaneously target all apple DAM and SVP genes in order to study their role and mode of action in the regulation of bud dormancy, budbreak and flowering. A synthetic construct carrying a hairpin fragment assembled from sequences specific to coding regions of three DAM and two SVP genes was used to generate transgenic lines. Reduced expression of DAM/SVP genes resulted in delayed leaf senescence and abscission in autumn, failure to enter bud dormancy in winter and continual growth of new leaves regardless of the season for over 3 years. Precocious flowering but normal flower morphology, fertility and fruit development were observed. The non-dormant phenotype was associated with modified phytohormone composition. The content of gibberellins (GAs) and jasmonates (JAs) was significantly increased in terminal buds of RNAi lines compared with wildtype plants, accompanied by elevated expression of the key GA biosynthesis pathway gene GIBBERELLIN 20 OXIDASE-2 (MdGA20ox-2) along with the FLOWERING LOCUS T gene MdFT2. The key mediator of plasmodesmatal closure, MdCALLOSE SYNTHASE 1 (MdCALS1), was repressed in RNAi lines. This study provides functional evidence for the role of DAM/SVP genes in vegetative phenology of apple and paves the way for production of low-chill varieties suitable for growth in warming climates.

Biosynthesis of the Dihydrochalcone Sweetener Trilobatin Requires Phloretin Glycosyltransferase2.

Epidemics of obesity and type 2 diabetes drive strong consumer interest in plant-based low-calorie sweeteners. Trilobatin is a sweetener found at high concentrations in the leaves of a range of crabapple (Malus) species, but not in domesticated apple (Malus × domestica) leaves, which contain trilobatin's bitter positional isomer phloridzin. Variation in trilobatin content was mapped to the Trilobatin locus on LG 7 in a segregating population developed from a cross between domesticated apples and crabapples. Phloretin glycosyltransferase2 (PGT2) was identified by activity-directed protein purification and differential gene expression analysis in samples high in trilobatin but low in phloridzin. Markers developed for PGT2 cosegregated strictly with the Trilobatin locus. Biochemical analysis showed PGT2 efficiently catalyzed 4'-o-glycosylation of phloretin to trilobatin as well as 3-hydroxyphloretin to sieboldin. Transient expression of double bond reductase, chalcone synthase, and PGT2 genes reconstituted the apple pathway for trilobatin production in Nicotiana benthamiana Transgenic M. × domestica plants overexpressing PGT2 produced high concentrations of trilobatin in young leaves. Transgenic plants were phenotypically normal, and no differences in disease susceptibility were observed compared to wild-type plants grown under simulated field conditions. Sensory analysis indicated that apple leaf teas from PGT2 transgenics were readily discriminated from control leaf teas and were perceived as significantly sweeter. Identification of PGT2 allows marker-aided selection to be developed to breed apples containing trilobatin, and for high amounts of this natural low-calorie sweetener to be produced via biopharming and metabolic engineering in yeast.

Overexpression of chalcone isomerase in apple reduces phloridzin accumulation and increases susceptibility to herbivory by two-spotted mites.

Apples (Malus spp.) accumulate significant quantities of the dihydrochalcone glycoside, phloridzin, whilst pears (Pyrus spp.) do not. To explain this difference, we hypothesized that a metabolic bottleneck in the phenylpropanoid pathway might exist in apple. Expression analysis indicated that transcript levels of early phenylpropanoid pathway genes in apple and pear leaves were similar, except for chalcone isomerase (CHI), which was much lower in apple. Apples also showed very low CHI activity compared with pear. To relieve the bottleneck at CHI, transgenic apple plants overexpressing the Arabidopsis AtCHI gene were produced. Unlike other transgenic apples where phenylpropanoid flux was manipulated, AtCHI overexpression (CHIox) plants were phenotypically indistinguishable from wild-type, except for an increase in red pigmentation in expanding leaves. CHIox plants accumulated slightly increased levels of flavanols and flavan-3-ols in the leaves, but the major change was a 2.8- to 19-fold drop in phloridzin concentrations compared with wild-type. The impact of these phytochemical changes on insect preference was studied using a two-choice leaf assay with the polyphagous apple pest, the two-spotted spider mite (Tetranychus urticae Koch). Transgenic CHIox leaves were more susceptible to herbivory, an effect that could be reversed (complemented) by application of phloridzin to transgenic leaves. Taken together, these findings shed new light on phenylpropanoid biosynthesis in apple and suggest a new physiological role for phloridzin as an antifeedant in leaves.

Apple B-box factors regulate light-responsive anthocyanin biosynthesis genes.

Environmentally-responsive genes can affect fruit red colour via the activation of MYB transcription factors. The apple B-box (BBX) gene, BBX33/CONSTANS-like 11 (COL11) has been reported to influence apple red-skin colour in a light- and temperature-dependent manner. To further understand the role of apple BBX genes, other members of the BBX family were examined for effects on colour regulation. Expression of 23 BBX genes in apple skin was analysed during fruit development. We investigated the diurnal rhythm of expression of the BBX genes, the anthocyanin biosynthetic genes and a MYB activator, MYB10. Transactivation assays on the MYB10 promoter, showed that BBX proteins 1, 17, 15, 35, 51, and 54 were able to directly function as activators. Using truncated versions of the MYB10 promoter, a key region was identified for activation by BBX1. BBX1 enhanced the activation of MYB10 and MdbHLH3 on the promoter of the anthocyanin biosynthetic gene DFR. In transformed apple lines, over-expression of BBX1 reduced internal ethylene content and altered both cyanidin concentration and associated gene expression. We propose that, along with environmental signals, the control of MYB10 expression by BBXs in 'Royal Gala' fruit involves the integration of the expression of multiple BBXs to regulate fruit colour.

Genetic control of α-farnesene production in apple fruit and its role in fungal pathogenesis.

Terpenes are important compounds in plant trophic interactions. A meta-analysis of GC-MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)-α-farnesene. Four quantitative trait loci (QTLs) for α-farnesene production in ripe fruit were identified in a segregating 'Royal Gala' (RG) × 'Granny Smith' (GS) population with one major QTL on linkage group 10 co-locating with the MdAFS1 (α-farnesene synthase-1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC-MS analysis of headspace and solvent-extracted terpenes showing that cold-treated GS apples produced higher levels of (E,E)-α-farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)-α-farnesene. To evaluate the role of (E,E)-α-farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post-harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)-α-farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post-inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)-α-farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.

Expression of MdCCD7 in the scion determines the extent of sylleptic branching and the primary shoot growth rate of apple trees.

Branching has a major influence on the overall shape and productivity of a plant. Strigolactones (SLs) have been identified as plant hormones that have a key role in suppressing the outgrowth of axillary meristems. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes are integral to the biosynthesis of SLs and are well characterized in annual plants, but their role in woody perennials is relatively unknown. We identified CCD7 and CCD8 orthologues from apple and demonstrated that MdCCD7 and MdCCD8 are able to complement the Arabidopsis branching mutants max3 and max4 respectively, indicating conserved function. RNAi lines of MdCCD7 show reduced gene expression and increased branching in apple. We performed reciprocal grafting experiments with combinations of MdCCD7 RNAi and wild-type 'Royal Gala' as rootstocks and scion. Unexpectedly, wild-type roots were unable to suppress branching in MdCCD7 RNAi scions. Another key finding was that MdCCD7 RNAi scions initiated phytomers at an increased rate relative to the wild type, resulting in a greater node number and primary shoot length. We suggest that localized SL biosynthesis in the shoot, rather than roots, controls axillary bud outgrowth and shoot growth rate in apple.

Ectopic expression of the PISTILLATA homologous MdPI inhibits fruit tissue growth and changes fruit shape in apple.

Fruit shape represents a key trait that consumers use to identify and select preferred cultivars, and although the manipulation of this trait is an opportunity to create novel, differentiated products, the molecular mechanisms regulating fruit shape are poorly understood in tree fruits. In this study, we have shown that ectopic expression of Malus domestica PISTILLATA (MdPI), the apple ortholog of the floral organ identity gene PISTILLATA (PI), regulates apple fruit tissue growth and shape. MdPI is a single-copy gene, and its expression is high during flower development but barely detectable soon after pollination. Transgenic apple plants with ectopic expression of MdPI produced flowers with white sepals and a conversion of sepals to petals. Interestingly, these plants produced distinctly flattened fruit as a consequence of reduced cell growth at the basipetal position of the fruit. These altered sepal and fruit phenotypes have not been observed in studies using Arabidopsis. This study using apple has advanced our understanding of PI functions outside the control of petal and stamen identity and provided molecular genetic information useful for manipulating fruit tissue growth and fruit shape.

Multiple Copies of a Simple MYB-Binding Site Confers Trans-regulation by Specific Flavonoid-Related R2R3 MYBs in Diverse Species.

In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of MYB10 in pear (PcMYB10) and Arabidopsis (AtMY75) elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3'5'H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants.

Silencing a phloretin-specific glycosyltransferase perturbs both general phenylpropanoid biosynthesis and plant development.

The polyphenol profile of apple (Malus × domestica) is dominated by the dihydrochalcone glycoside phloridzin, but its physiological role is yet to be elucidated. Biosynthesis of phloridzin occurs as a side branch of the main phenylpropanoid pathway, with the final step mediated by the phloretin-specific glycosyltransferase UGT88F1. Unexpectedly, given that UGTs are sometimes viewed as 'decorating enzymes', UGT88F1 knockdown lines were severely dwarfed, with greatly reduced internode lengths, narrow lanceolate leaves, and changes in leaf and fruit cellular morphology. These changes suggested that auxin transport had been altered in the knockdown lines, which was confirmed in assays showing that auxin flux from the shoot apex was increased in the transgenic lines. Metabolite analysis revealed no accumulation of the phloretin aglycone, as well as decreases in many non-target phenylpropanoid compounds. This decreased accumulation of metabolites appeared to be mediated by the repression of the phenylpropanoid pathway via a reduction in key transcript levels (e.g. phenylalanine ammonia lyase, PAL) and enzyme activities (PAL and chalcone synthase). Application of exogenous phloridzin to the UGT88F1 knockdown lines in tissue culture enhanced axial leaf growth and partially restored some aspects of 'normal' apple leaf growth. Together, our results strongly implicate dihydrochalcones as critical compounds in modulating phenylpropanoid pathway flux and establishing auxin patterning early in apple development.

SVP-like MADS Box Genes Control Dormancy and Budbreak in Apple.

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 PHASE-like (MdSVPs) genes from apple (Malus × 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.

Alcohol acyl transferase 1 links two distinct volatile pathways that produce esters and phenylpropenes in apple fruit.

Fruit accumulate a diverse set of volatiles including esters and phenylpropenes. Volatile esters are synthesised via fatty acid degradation or from amino acid precursors, with the final step being catalysed by alcohol acyl transferases (AATs). Phenylpropenes are produced as a side branch of the general phenylpropanoid pathway. Major quantitative trait loci (QTLs) on apple (Malus × domestica) linkage group (LG)2 for production of the phenylpropene estragole and volatile esters (including 2-methylbutyl acetate and hexyl acetate) both co-located with the MdAAT1 gene. MdAAT1 has previously been shown to be required for volatile ester production in apple (Plant J., 2014, https://doi.org/10.1111/tpj.12518), and here we show it is also required to produce p-hydroxycinnamyl acetates that serve as substrates for a bifunctional chavicol/eugenol synthase (MdoPhR5) in ripe apple fruit. Fruit from transgenic 'Royal Gala' MdAAT1 knockdown lines produced significantly reduced phenylpropene levels, whilst manipulation of the phenylpropanoid pathway using MdCHS (chalcone synthase) knockout and MdMYB10 over-expression lines increased phenylpropene production. Transient expression of MdAAT1, MdoPhR5 and MdoOMT1 (O-methyltransferase) genes reconstituted the apple pathway to estragole production in tobacco. AATs from ripe strawberry (SAAT1) and tomato (SlAAT1) fruit can also utilise p-coumaryl and coniferyl alcohols, indicating that ripening-related AATs are likely to link volatile ester and phenylpropene production in many different fruit.

How microRNA172 affects fruit growth in different species is dependent on fruit type.

microRNA172 (miR172) expression has been shown to have a positive effect on Arabidopsis fruit (siliques) growth. In contrast, over-expression of miR172 has a negative influence on fruit growth in apple, resulting in a dramatic reduction in fruit size. This negative influence is supported by the results of analyzing a transposable element (TE) insertional allele of a MIR172 gene that has reduced expression of the miRNA and is associated with an increase in fruit size. Arabidopsis siliques are a dry fruit derived from ovary tissues, whereas apple is a fleshy pome fruit derived mostly from hypanthium tissues. A model has been developed to explain the contrasting impact of miR172 expression in these two plant species based on the differences in their fruit structure. Transgenic apple plants with extremely high levels of miR172 overexpression produced flowers consisting of carpel tissues only, which failed to produce fruit. By comparison, in tomato, a fleshy berry fruit derived from the ovary, high level over-expression of the same miR172 resulted in carpel-only flowers which developed into parthenocarpic fruit. These results further indicate that the influence of miR172 on fruit growth in different plant species depends on its fruit type.

A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution.

The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of a microRNA gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over-expression of miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co-located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops.

The O-methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit.

Phenylpropenes, such as eugenol and trans-anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non-producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co-located with seven candidate genes for phenylpropene O-methyltransferases (MdoOMT1-7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over-expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.