Identification of genes associated with the regulation of cold tolerance and the RNA movement in the grafted apple.

In grafted apple, rootstock-derived signals influence scion cold tolerance by initiating physiological changes to survive over the winter. To understand the underlying molecular interactions between scion and rootstock responsive to cold, we developed transcriptomics and metabolomics data in the stems of two scion/rootstock combinations, 'Gala'/'G202' (cold resistant rootstock) and 'Gala'/'M9' (cold susceptible rootstock). Outer layers of scion and rootstock stem, including vascular tissues, were collected from the field-grown grafted apple during the winter. The clustering of differentially expressed genes (DEGs) and gene ontology enrichment indicated distinct expression dynamics in the two graft combinations, which supports the dependency of scion cold tolerance on the rootstock genotypes. We identified 544 potentially mobile mRNAs of DEGs showing highly-correlated seasonal dynamics between scion and rootstock. The mobility of a subset of 544 mRNAs was validated by translocated genome-wide variants and the measurements of selected RNA mobility in tobacco and Arabidopsis. We detected orthologous genes of potentially mobile mRNAs in Arabidopsis thaliana, which belong to cold regulatory networks with RNA mobility. Together, our study provides a comprehensive insight into gene interactions and signal exchange between scion and rootstock responsive to cold. This will serve for future research to enhance cold tolerance of grafted tree crops.

Effects of Daily Ingestion of Two SunGold Kiwifruit for 6 Weeks on Metabolic and Inflammatory Biomarkers: A Randomized, Cross-Over, Exploratory Intervention Study.

Kiwifruit contain many components, some considered beneficial, such as vitamins, phytochemicals and dietary fibre, and others potentially harmful, such as fructose and glucose in fruit sugars. In a 6-week, randomised, crossover study aimed at exploring the net effects of daily consumption of kiwifruit, 23 healthy participants consumed two Actinidia chinensis var. chinensis 'Zesy002' (marketed as Zespri™ SunGold™ Kiwifruit) per day as part of their customary diet (intervention) or without kiwifruit (control) as their customary diet for 6 weeks in a cross-over study. Anthropometric data, venous blood, and urine samples were collected at the start and end of the 6-week intervention and control periods for the measurement of physical changes, plasma glucose, insulin, glycated haemoglobin, short-chain fatty acids, blood lipids, uric acid, inflammatory biomarkers, and urinary ascorbic acid. Variables were measured between the start and finish of interventions, and between intervention and control periods. Food diaries were completed on the 3 days before blood sampling to estimate dietary ascorbic acid and dietary fibre intakes. Despite urinary vitamin C and food diaries indicating compliance, and good precision in measurements, there were no appreciable changes in biomarkers during the study, either within or between intervention and control periods, that would indicate a change in health status. Thus, the sizes of any effects of kiwifruit ingestion were too small to become significant under the test conditions used, indicating a high probability that daily ingestion of two SunGold kiwifruit is safe with respect to metabolic health.

GWAS provides new insights into the genetic mechanisms of phytochemicals production and red skin colour in apple.

Understanding the genetic architecture of apple phytochemicals, and their interplay with conventional selection traits, is critical for the development of new apple cultivars with enhanced health benefits. Apple accessions (n = 344) used for this genome-wide association study (GWAS) represented the wide diversity of metabolic profiles in the domesticated and wild Malus genepools. Fruit samples were phenotyped for 34 metabolites, including a stable vitamin C glycoside "ascorbic acid 2-ß-glucoside" (AA-2ßG), and the accessions were genotyped using the Apple 20 K SNP Array. Several fruit quality traits, including red skin over-colour (OCOL), were also assessed. Wild Malus accessions showed at least 2-fold higher average content of several metabolites (e.g. ascorbic acid, chlorogenic acid, phloridzin, and trilobatin) than Malus domestica accessions. Several new genomic regions and potential candidate genes underpinning the genetic diversity of apple phytochemicals were identified. The percentage of phenotypic variance explained by the best SNP ranged between 3% and 21% for the different metabolites. Novel association signals for OCOL in the syntenic regions on chromosomes 13 and 16 suggested that whole genome duplication has played a role in the evolution of apple red skin colour. Genetic correlations between phytochemicals and sensory traits were moderate. This study will assist in the selection of Malus accessions with specific phytochemical profiles to establish innovative genomics-based breeding strategies for the development of apple cultivars with enhanced nutritional value.

High-density linkage map construction in an autotetraploid blueberry population and detection of quantitative trait loci for anthocyanin content.

Highbush blueberry (Vaccinium corymbosum, 2n = 4x = 48) is the most cultivated type of blueberry, both in New Zealand and overseas. Its perceived nutritional value is conferred by phytonutrients, particularly anthocyanins. Identifying the genetic mechanisms that control the biosynthesis of these metabolites would enable faster development of cultivars with improved fruit qualities. Here, we used recently released tools for genetic mapping in autotetraploids to build a high-density linkage map in highbush blueberry and to detect quantitative trait loci (QTLs) for fruit anthocyanin content. Genotyping was performed by target sequencing, with ∼18,000 single nucleotide polymorphism (SNP) markers being mapped into 12 phased linkage groups (LGs). Fruits were harvested when ripe for two seasons and analyzed with high-performance liquid chromatography-mass spectrometry (HPLC-MS): 25 different anthocyanin compounds were identified and quantified. Two major QTLs that were stable across years were discovered, one on LG2 and one on LG4, and the underlying candidate genes were identified. Interestingly, the presence of anthocyanins containing acylated sugars appeared to be under strong genetic control. Information gained in this study will enable the design of molecular markers for marker-assisted selection and will help build a better understanding of the genetic control of anthocyanin biosynthesis in this crop.

Flavonoid biosynthesis is differentially altered in detached and attached ripening bilberries in response to spectral light quality.

Light spectral quality is known to affect flavonoid biosynthesis during fruit ripening. However, the response of fruits to different light conditions, when ripening autonomously from the parent plant (detached), has been less explored. In this study, we analyzed the effect of light quality on detached and naturally ripening (attached) non-climacteric wild bilberry (Vaccinium myrtillus L.) fruits accumulating high amounts of anthocyanins and flavonols. Our results indicated contrasting responses for the accumulation of phenolic compounds in the berries in response to red and blue light treatments. For detached berries, supplemental blue light resulted in the highest accumulation of anthocyanins, while naturally ripening berries had elevated accumulation under supplemental red light treatment. Both red and blue supplemental light increased the expression levels of all the major structural genes of the flavonoid pathway during ripening. Notably, the key regulatory gene of anthocyanin biosynthesis, VmMYBA1, was found to express fivefold higher under blue light treatment in the detached berries compared to the control. The red light treatment of naturally ripening berries selectively increased the delphinidin branch of anthocyanins, whereas in detached berries, blue light increased other anthocyanin classes along with delphinidins. In addition, red and far-red light had a positive influence on the accumulation of flavonols, especially quercetin and myricetin glycoside derivatives, in both ripening conditions. Our results of differential light effects on attached and detached berries, which lacks signaling from the mother plant, provide new insights in understanding the light-mediated regulatory mechanisms in non-climacteric fruit ripening.

Influence of oral administration of kukoamine A on blood pressure in a rat hypertension model.

The benefits of lowering blood pressure (BP) are well established for the prevention of cardiovascular disease. While there are a number of pharmaceuticals available for lowering BP, there is considerable interest in using dietary modifications, lifestyle and behaviour changes as alternative strategies. Kukoamines, caffeic acid derivatives of polyamines present in solanaceous plants, have been reported to reduce BP. We investigated the effect of orally administered synthetic kukoamine A on BP in the Spontaneously Hypertensive Rat (SHR) laboratory animal model of hypertension. Prior to the hypertension study, we determined the safety of the synthetic kukoamine A in a single oral dose (5 or 10 mg kg-1 bodyweight) 14-day observational study in mice. No negative effects of the oral administration of kukoamine A were observed. We subsequently investigated the effect of daily oral doses of kukoamine A (0, 5, 10 mg kg-1 bodyweight) for 35 days using the SHR rat model of hypertension. The normotensive control Wistar Kyoto (WKY) strain was used to provide a baseline for normal BP in rats. We observed no effect of orally administered synthetic kukoamine A on arterial hypertension in this laboratory animal model of hypertension.

Resolving the developmental distribution patterns of polyphenols and related primary metabolites in bilberry (Vaccinium myrtillus) fruit.

Bilberry (Vaccinium myrtillus) is a commercially important wild berry species, which accumulates high amounts of polyphenols, particularly anthocyanins, in the skin and flesh. Whilst a number of studies have quantified these phytochemicals in intact ripe bilberry fruit, we extend the current knowledge by investigating the spatial distribution of anthocyanin-associated polyphenols in fruit tissue, and study their links with primary metabolism during ripening. To address this, we used LC-MS and mass spectrometry imaging to measure and map primary and secondary metabolites in fruit. Correlation analysis showed that five sugars displayed strong positive correlations with anthocyanin accumulation, whereas all amino acids were negatively correlated. The accumulation patterns of polyphenols correlated in fruit skin and flesh, but altered with development. Finally, spatial segmentation analysis revealed that the chemical signatures of ripening first appear at defined regions under the skin and rapidly expand to encompass the entire fruit at the eating-ripe stage.

Onco-Preventive and Chemo-Protective Effects of Apple Bioactive Compounds.

Cancer is one of the leading causes of death globally. Epidemiological studies have strongly linked a diet high in fruits to a lower incidence of cancer. Furthermore, extensive research shows that secondary plant metabolites known as phytochemicals, which are commonly found in fruits, have onco-preventive and chemo-protective effects. Apple is a commonly consumed fruit worldwide that is available all year round and is a rich source of phytochemicals. In this review, we summarize the association of apple consumption with cancer incidence based on findings from epidemiological and cohort studies. We further provide a comprehensive review of the main phytochemical patterns observed in apples and their bioavailability after consumption. Finally, we report on the latest findings from in vitro and in vivo studies highlighting some of the key molecular mechanisms targeted by apple phytochemicals in relation to inhibiting multiple 'hallmarks of cancer' that are important in the progression of cancer.

Kiwifruit Metabolomics-An Investigation of within Orchard Metabolite Variability of Two Cultivars of Actinidia chinensis.

Plant metabolomics within field-based food production systems is challenging owing to environmental variability and the complex architecture and metabolic growth cycles of plants. Kiwifruit cultivars of Actinidia chinensis are vigorous perennial vines grown as clones in highly structured orchard environments, intensively managed to maximize fruit yield and quality. To understand the metabolic responses of vines to orchard management practices, we needed to better understand the various sources of metabolic variability encountered in the orchard. Triplicate composite leaf, internode and fruit (mature and immature) samples were collected from each of six Actinidia chinensis var. deliciosa 'Hayward' and A. chinensis var. chinensis 'Zesy002' kiwifruit vines at three times during the growing season and measured by LC-MS. In general, there was more variation in metabolite concentrations within vines than between vines, with 'Hayward' showing a greater percentage of within-vine variability than 'Zesy002' (c. 90 vs. 70% respectively). In specific tissues, the sampler, infection by Pseudomonas syringae var. actinidiae and the rootstock also influenced metabolite variability. A similar pattern of metabolic variability was observed from quantitative analysis of specific carbohydrates and phytohormones. High within-vine metabolic variability indicates that it is more important to obtain sufficient replicate samples than to sample from multiple vines. These data provide an objective basis for optimizing metabolite sampling strategies within kiwifruit orchards.

MYBA and MYBPA transcription factors co-regulate anthocyanin biosynthesis in blue-coloured berries.

The regulatory network of R2R3 MYB transcription factors in anthocyanin biosynthesis is not fully understood in blue-coloured berries containing delphinidin compounds. We used blue berries of bilberry (Vaccinium myrtillus) to comprehensively characterise flavonoid-regulating R2R3 MYBs, which revealed a new type of co-regulation in anthocyanin biosynthesis between members of MYBA-, MYBPA1- and MYBPA2-subgroups. VmMYBA1, VmMYBPA1.1 and VmMYBPA2.2 expression was elevated at berry ripening and by abscisic acid treatment. Additionally, VmMYBA1 and VmMYBPA1.1 expression was strongly downregulated in a white berry mutant. Complementation and transient overexpression assays confirmed VmMYBA1 and VmMYBA2 to induce anthocyanin accumulation. Promoter activation assays showed that VmMYBA1, VmMYBPA1.1 and VmMYBPA2.2 had similar activity towards dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), but differential regulation activity for UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) and flavonoid 3'5'-hydroxylase (F3'5'H) promoters. Silencing of VmMYBPA1.1 in berries led to the downregulation of key anthocyanin and delphinidin biosynthesis genes. Functional analyses of other MYBPA regulators, and a member of novel MYBPA3 subgroup, associated them with proanthocyanidin biosynthesis and F3'5'H expression. The existence of 18 flavonoid-regulating MYBs indicated gene duplication, which may have enabled functional diversification among MYBA, MYBPA1 and MYBPA2 subgroups. Our results provide new insights into the intricate regulation of the complex anthocyanin profile found in blue-coloured berries involving regulation of both cyanidin and delphinidin branches.

Production of Betacyanins in Transgenic Nicotiana tabacum Increases Tolerance to Salinity.

Although red betalain pigments (betacyanins) have been associated with salinity tolerance in some halophytes like Disphyma australe, efforts to determine whether they have a causal role and the underlying mechanisms have been hampered by a lack of a model system. To address this, we engineered betalain-producing Nicotiana tabacum, by the introduction of three betalain biosynthetic genes. The plants were violet-red due to the accumulation of three betacyanins: betanin, isobetanin, and betanidin. Under salt stress, betacyanic seedlings had increased survivability and leaves of mature plants had higher photochemical quantum yields of photosystem II (F v /F m ) and faster photosynthetic recovery after saturating light treatment. Under salt stress, compared to controls betacyanic leaf disks had no loss of carotenoids, a slower rate of chlorophyll degradation, and higher F v /F m values. Furthermore, simulation of betacyanin pigmentation by using a red filter cover improved F v /F m value of green tissue under salt stress. Our results confirm a direct causal role of betacyanins in plant salinity tolerance and indicate a key mechanism is photoprotection. A role in delaying leaf senescence was also indicated, and the enhanced antioxidant capability of the betacyanic leaves suggested a potential contribution to scavenging reactive oxygen species. The study can inform the development of novel biotechnological approaches to improving agricultural productivity in saline-affected areas.

Cold stress triggers premature fruit abscission through ABA-dependent signal transduction in early developing apple.

Fruit abscission is a complex physiological process that is regulated by internal and environmental factors. During early development, apple fruit are exposed to extreme temperature fluctuations that are associated with premature fruit drop; however, their effect on fruit abscission is largely unknown. We hypothesized that fruit abscission is triggered by cold stress and investigated the molecular basis of premature fruit drop using RNA-Seq and metabolomics data from apple fruit undergoing abscission following cold stress in the field. Genes responsive to abscisic acid signaling and cell wall degradation were upregulated during abscission, consistent with the increased abscisic acid concentrations detected by liquid chromatography-mass spectrometry. We performed ex vivo cold shock experiments with excised tree subunits consisting of a branch, pedicel, and fruit. Abscission induction occurred in the cold-stressed subunits with concurrent upregulation of abscisic acid biosynthesis (MdNCED1) and metabolism (MdCYP707A) genes, and ethylene biosynthesis (MdACS1) and receptor (MdETR2) genes in the pedicel. Another key finding was the activation of cytoplasmic streaming in abscission-zone cells detected by electron microscopy. Our results provide a novel insight into the molecular basis of fruit abscission physiology in response to cold stress in apple.

A Polyphenol Enriched Variety of Apple Alters Circulating Immune Cell Gene Expression and Faecal Microbiota Composition in Healthy Adults: A Randomized Controlled Trial.

Polyphenols within fruits and vegetables may contribute to health benefits due to their consumption, with the anthocyanin sub-set also adding colour. The Lemonade™ apple variety has green skin and white flesh, with low anthocyanin content, while some apple varieties have high anthocyanin content in both the skin and flesh. Effects of red compared with white-fleshed apples were studied in healthy human subjects in a randomized, placebo-controlled, cross-over intervention trial. Twenty-five healthy subjects consumed dried daily portions of the red-fleshed or placebo (white-fleshed) apple for two weeks, followed by one-week washout and further two-week crossover period. During the study, volunteers provided faecal samples for microbiota composition analysis and blood samples for peripheral blood mononuclear cell (PBMC) gene expression analysis. Subtle differences were observed in the faecal microbiota of subjects that were fed the different apples, with significant (p < 0.05) reductions in relative abundances of Streptococcus, Ruminococcus, Blautia, and Roseburia, and increased relative abundances of Sutterella, Butyricicoccus, and Lactobacillus in subjects after consuming the red apple. Changes in PBMC gene expression showed 18 mRNA transcripts were differentially expressed between the two groups, of which 16 were immunoglobulin related genes. Pathway analysis showed that these genes had roles in pathways such as immunoglobulin production, B cell-mediated immunity, complement activation, and phagocytosis. In conclusion, this study shows that anthocyanin-rich apples may influence immune function compared to control apples, with changes potentially associated with differences in the faecal microbiota.

2-O-ß-d-Glucopyranosyl l-Ascorbic Acid, a Stable Form of Vitamin C, Is Widespread in Crop Plants.

2-O-ß-d-Glucopyranosyl l-ascorbic acid (AA-2ßG) is a stable, bioavailable vitamin C (AA) derivative. We report the distribution and seasonal variation of AA-2ßG in apples and its occurrence in other domesticated crops and in wild harvested Ma̅ori foods. Liquid chromatography-mass spectrometry analyses showed high AA-2ßG concentrations in crab apples (Malus sylvestris) but low concentrations in domesticated apples. Leaves of crab and domesticated apple cultivars contained similar intermediate AA-2ßG concentrations. Fruits and leaves of other crops were analyzed: mainly Rosaceae but also Actinidiaceae and Ericaceae. AA-2ßG was detected in all leaves (0.5-6.1 mg/100 g fr. wt.) but was at lower concentrations in most fruits (0.0-0.5 mg/100 g fr. wt.) except for crab apples (79.4 mg/100 g fr. wt.). Ma̅ori foods from Solanaceae, Piperaceae, Asteraceae, and a fern of Aspleniaceae also contained AA-2ßG. This extensive occurrence suggests a general role in AA metabolism for AA-2ßG.

Spatiotemporal Modulation of Flavonoid Metabolism in Blueberries.

Blueberries are distinguished by their purple-blue fruit color, which develops during ripening and is derived from a characteristic composition of flavonoid-derived anthocyanin pigments. The production of anthocyanins is confined to fruit skin, leaving the colorless fruit flesh devoid of these compounds. By linking accumulation patterns of phenolic metabolites with gene transcription in Northern Highbush (Vaccinium corymbosum) and Rabbiteye (Vaccinium virgatum) blueberry, we investigated factors limiting anthocyanin production in berry flesh. We find that flavonoid production was generally lower in fruit flesh compared with skin and concentrations further declined during maturation. A common set of structural genes was identified across both species, indicating that tissue-specific flavonoid biosynthesis was dependent on co-expression of multiple pathway genes and limited by the phenylpropanoid pathway in combination with CHS, F3H, and ANS as potential pathway bottlenecks. While metabolite concentrations were comparable between the blueberry genotypes when fully ripe, the anthocyanin composition was distinct and depended on the degree of hydroxylation/methoxylation of the anthocyanidin moiety in combination with genotype-specific glycosylation patterns. Co-correlation analysis of phenolic metabolites with pathway structural genes revealed characteristic isoforms of O-methyltransferases and UDP-glucose:flavonoid-3-O-glycosyltransferase that were likely to modulate anthocyanin composition. Finally, we identified candidate transcriptional regulators that were co-expressed with structural genes, including the activators MYBA, MYBPA1, and bHLH2 together with the repressor MYBC2, which suggested an interdependent role in anthocyanin regulation.

Discovery of a stable vitamin C glycoside in crab apples (Malus sylvestris).

Non-targeted LC-MS metabolomics on fruit of three wild and domesticated apple species (Malus sylvestris, M. sieversii and M. domestica) showed that two crab apple (M. sylvestris) accessions were distinguished by high concentrations of an ascorbic acid glycoside (AAG). This was partly purified, but key NMR signals were masked by inseparable sucrose. Reference samples of 2-O-ß-D-glucopyranosyl L-ascorbic acid and 2-O-ß-D-galactopyranosyl L-ascorbic acid were synthesised, but both coincided with the crab apple AAG on LC-MS. Peracetylation of the crab apple extract allowed both purification and characterisation, and the AAG was proven to be 2-O-ß-D-glucopyranosyl L-ascorbic acid by comparison of 1H NMR, HRMS and HPLC data with synthesised peracetylated ascorbyl glycoside standards. The stability of the natural AA 2-ß-glycoside was similar to synthetic 2-O-α-D-glucopyranosyl L-ascorbic acid, used widely in cosmetic and pharmaceutical products. This discovery in crab apples (Rosaceae) is only the fourth reported occurrence of any ascorbyl glycoside from plants, the others being from Cucurbitaceae, Solanaceae and Brassicaceae. It is hypothesised that AAGs may be more widespread in plants than currently realised.

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.

Carbon starvation reduces carbohydrate and anthocyanin accumulation in red-fleshed fruit via trehalose 6-phosphate and MYB27.

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel-coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red-fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6-phosphate. RNA-Seq analysis showed down-regulation of both gene-encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6-phosphate synthase gene TPS1.1a was down-regulated, whereas putative regulatory TPS7 and TPS11 were strongly up-regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up-regulated, whereas MYB27 was also up-regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6-phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits.

Quantitative trait loci influencing pentacyclic triterpene composition in apple fruit peel.

The chemical composition of pentacyclic triterpenes was analysed using a 'Royal Gala' x 'Granny Smith' segregating population in 2013 and 2015, using apple peels extracted from mature fruit at harvest and after 12 weeks of cold storage. In 2013, 20 compound isoforms from nine unique compound classes were measured for both treatments. In 2015, 20 and 17 compound isoforms from eight unique compound classes were measured at harvest and after cold storage, respectively. In total, 68 quantitative trait loci (QTLs) were detected on 13 linkage groups (LG). Thirty two and 36 QTLs were detected for compounds measured at harvest and after cold storage, respectively. The apple chromosomes with the most QTLs were LG3, LG5, LG9 and LG17. The largest effect QTL was for trihydroxy-urs-12-ene-28-oic acid, located on LG5; this was measured in 2015 after storage, and was inherited from the 'Royal Gala' parent (24.9% of the phenotypic variation explained).

Red to Brown: An Elevated Anthocyanic Response in Apple Drives Ethylene to Advance Maturity and Fruit Flesh Browning.

The elevation of anthocyanin contents in fruits and vegetables is a breeding target for many crops. In some fruit, such as tomato, higher anthocyanin concentrations enhance storage and shelf life. In contrast, highly anthocyanic red-fleshed apples (Malus x domestica) have an increased incidence of internal browning flesh disorder (IBFD). To determine the mechanisms underlying this, 'Royal Gala' cultivar apples over-expressing the anthocyanin-related transcription factor (TF) MYB10 (35S:MYB10), which produces fruit with highly pigmented flesh, were compared with standard 'Royal Gala' Wild Type (WT) grown under the same conditions. We saw no incidence of IBFD in WT 'Royal Gala' but the over-expression of MYB10 in the same genetic background resulted in a high rate of IBDF. We assessed concentrations of potential substrates for IBDF and a comparison of metabolites in these apples showed that anthocyanins, chlorogenic acid, pro-cyanidins, flavon-3-ols, and quercetin were all higher in the MYB10 lines. For the flavol-3-ols sub-group, epicatechin rather than catechin was elevated in MYB10 lines compared with the control fruit. Internal ethylene concentrations were measured throughout fruit development and were significantly higher in 35S:MYB10 lines, and ethylene was detected at an earlier developmental stage pre-harvest. Expression analysis of key genes associated with ethylene biosynthesis (aminocyclopropane-1-carboxylic acid synthase and oxidase; ACS and ACO) and polyphenol oxidase (PPO) showed the potential for increased ethylene production and the mechanism for enhanced PPO-mediated browning. The expression of a transcription factor of the ethylene response factor (ERF) class, ERF106, was elevated in red flesh. Analysis of transcriptional activation by MYB10 showed that this transcription factor could activate the expression of apple ACS, ACO, and ERF106 genes. Our data show a link between the elevation of anthocyanin-related transcription factors and an undesirable fruit disorder. The accelerated advancement of maturity via premature ethylene induction has implications for the breeding and storage of these more highly pigmented plant products.