Quantitation of circulating short-chain fatty acids in small volume blood samples from animals and humans.

BACKGROUND: The role of gut microbiota in human health has been intensively studied and more recently shifted from emphasis on composition towards function. Function is partly mediated through formed metabolites. Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate as well as their branched analogues represent major products from gut fermentation of dietary fibre and proteins, respectively. Robust and high-throughput analysis of SCFAs in small volume blood samples have proven difficult. Major obstacles come from the ubiquitous presence of SCFAs that leads to contaminations and unstable analytical results because of the high volatility of these small molecules. Comprehensive and comparable data on the variation of SCFAs in blood samples from different blood matrices and mammal species including humans is lacking. Therefore, our aim was to develop and evaluate a stable and robust method for quantitation of 8 SCFAs and related fermentation products in small volume blood plasma samples and to investigate their variation in humans and different animal species. RESULTS: Derivatization was a successful approach for measurement of SCFAs in biological samples but quenching of the derivatization reaction was crucial to obtain long-term stability of the derivatized analytes. In total 9 compounds (including succinic acid) were separated in 5 min. The method was linear over the range 0.6-3200 nM formic (FA), acetic (AA), 0.3-1600 nM propionic (PA), and 0.16-800 nM for butyric (BA)-, isobutyric (IBA)-, valeric (VA)-, isovaleric (IVA)-, succinic (SA) and caproic acid (CA). The precision ranged ≤12 % within days and ≤28 % between days (except for CA and VA) in three different plasma quality control (QC) samples (29 batches analyzed over 3 months). The extraction recovery was on average 94 % for the different SCFAs. Typical interquartile range (IQR) concentrations (µM) of SCFAs in human plasma samples were 168 µM (FA), 64 µM (AA), 2.2 µM (PA), 0.54 µM (BA), 0.66 µM (IBA), 0.18 µM (VA), 0.40 µM (IVA), and 0.34 µM (CA). In total, 55 samples per batch/day were successfully analyzed and in total 5380 human plasma samples measured over a 3-year timespan. SIGNIFICANCE: The developed UHPLC-MS based method was suitable for measuring SCFAs in small blood volume samples and enabled robust quantitative data.

Allelic variation of BBX24 is a dominant determinant controlling red coloration and dwarfism in pear.

Variation in anthocyanin biosynthesis in pear fruit provides genetic germplasm resources for breeding, while dwarfing is an important agronomic trait, which is beneficial to reduce the management costs and allow for the implementation of high-density cultivation. Here, we combined bulked segregant analysis (BSA), quantitative trait loci (QTL), and structural variation (SV) analysis to identify a 14-bp deletion which caused a frame shift mutation and resulted in the premature translation termination of a B-box (BBX) family of zinc transcription factor, PyBBX24, and its allelic variation termed PyBBX24ΔN14. PyBBX24ΔN14 overexpression promotes anthocyanin biosynthesis in pear, strawberry, Arabidopsis, tobacco, and tomato, while that of PyBBX24 did not. PyBBX24ΔN14 directly activates the transcription of PyUFGT and PyMYB10 through interaction with PyHY5. Moreover, stable overexpression of PyBBX24ΔN14 exhibits a dwarfing phenotype in Arabidopsis, tobacco, and tomato plants. PyBBX24ΔN14 can activate the expression of PyGA2ox8 via directly binding to its promoter, thereby deactivating bioactive GAs and reducing the plant height. However, the nuclear localization signal (NLS) and Valine-Proline (VP) motifs in the C-terminus of PyBBX24 reverse these effects. Interestingly, mutations leading to premature termination of PyBBX24 were also identified in red sports of un-related European pear varieties. We conclude that mutations in PyBBX24 gene link both an increase in pigmentation and a decrease in plant height.

Effects of Drought and Flooding on Phytohormones and Abscisic Acid Gene Expression in Kiwifruit.

Environmental extremes, such as drought and flooding, are becoming more common with global warming, resulting in significant crop losses. Understanding the mechanisms underlying the plant water stress response, regulated by the abscisic acid (ABA) pathway, is crucial to building resilience to climate change. Potted kiwifruit plants (two cultivars) were exposed to contrasting watering regimes (water logging and no water). Root and leaf tissues were sampled during the experiments to measure phytohormone levels and expression of ABA pathway genes. ABA increased significantly under drought conditions compared with the control and waterlogged plants. ABA-related gene responses were significantly greater in roots than leaves. ABA responsive genes, DREB2 and WRKY40, showed the greatest upregulation in roots with flooding, and the ABA biosynthesis gene, NCED3, with drought. Two ABA-catabolic genes, CYP707A i and ii were able to differentiate the water stress responses, with upregulation in flooding and downregulation in drought. This study has identified molecular markers and shown that water stress extremes induced strong phytohormone/ABA gene responses in the roots, which are the key site of water stress perception, supporting the theory kiwifruit plants regulate ABA to combat water stress.

Concentrations of Plasma Amino Acids and Neurotransmitters in Participants with Functional Gut Disorders and Healthy Controls.

Amino acids are important in several biochemical pathways as precursors to neurotransmitters which impact biological processes previously linked to functional gastrointestinal disorders (FGIDs). Dietary protein consumption, metabolic host processes, and the gut microbiome can influence the plasma concentration of amino acids and neurotransmitters, and their uptake by tissues. The aim of this analysis was to quantify 19 proteogenic and 4 non-proteogenic amino acids and 19 neurotransmitters (including precursors and catabolites, herein referred to as neurotransmitters) to ascertain if their circulating concentrations differed between healthy participants and those with FGIDs. Plasma proteogenic and non-proteogenic amino acids and neurotransmitters were measured using ultra-performance liquid chromatography and liquid chromatography-mass spectrometry, respectively, from 165 participants (Rome IV: irritable bowel syndrome (IBS-constipation, IBS-diarrhea), functional constipation, functional diarrhea, and healthy controls). There were significant differences (p < 0.05) in pairwise comparisons between healthy controls and specific FGID groups for branched-chain amino acids (BCAAs), ornithine, and alpha-aminobutyric acid. No other significant differences were observed for the neurotransmitters or any other amino acids analyzed. Multivariate and bivariate correlation analyses between proteogenic and non-proteogenic amino acids and neurotransmitters for constipation (constipation (IBS-C and functional constipation) and phenotypes diarrhea (IBS-D and functional diarrhea)) and healthy controls suggested that associations between BCAAs, 5-hydroxytryptophan, and kynurenine in combination with tyrosine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylacetic acid and associations with gamma-aminobutyric acid, glutamate, asparagine, and serine are likely disrupted in FGID phenotypes. In conclusion, although correlations were evident between some proteogenic and non-proteogenic amino acids and neurotransmitters, the results showed minor concentration differences in plasma proteogenic and non-proteogenic amino acids, amino acid-derived metabolites, and neurotransmitters between FGID phenotypes and healthy controls.

Two genes, ANS and UFGT2, from Vaccinium spp. are key steps for modulating anthocyanin production.

Anthocyanins are a major group of red to blue spectrum plant pigments with many consumer health benefits. Anthocyanins are derived from the flavonoid pathway and diversified by glycosylation and methylation, involving the concerted action of specific enzymes. Blueberry and bilberry (Vaccinium spp.) are regarded as 'superfruits' owing to their high content of flavonoids, especially anthocyanins. While ripening-related anthocyanin production in bilberry (V. myrtillus) and blueberry (V. corymbosum) is regulated by the transcriptional activator MYBA1, the role of specific structural genes in determining the concentration and composition of anthocyanins has not been functionally elucidated. We isolated three candidate genes, CHALCONE SYNTHASE (VmCHS1), ANTHOCYANIDIN SYNTHASE (VmANS) and UDP-GLUCOSE : FLAVONOID-3-O-GLYCOSYLTRANSFERASE (VcUFGT2), from Vaccinium, which were predominantly expressed in pigmented fruit skin tissue and showed high homology between bilberry and blueberry. Agrobacterium-mediated transient expression of Nicotiana benthamiana showed that overexpression of VcMYBA1 in combination with VmANS significantly increased anthocyanin concentration (3-fold). Overexpression of VmCHS1 showed no effect above that induced by VcMYBA1, while VcUFGT2 modulated anthocyanin composition to produce delphinidin-3-galactosylrhamnoside, not naturally produced in tobacco. In strawberry (Fragaria × ananassa), combined transient overexpression of VcUFGT2 with a FLAVONOID 3´,5´-HYDROXYLASE from kiwifruit (Actinidia melanandra) modulated the anthocyanin profile to include galactosides and arabinosides of delphinidin and cyanidin, major anthocyanins in blueberry and bilberry. These findings provide insight into the role of the final steps of biosynthesis in modulating anthocyanin production in Vaccinium and may contribute to the targeted breeding of new cultivars with improved nutritional properties.

Exogenous abscisic acid and sugar induce a cascade of ripening events associated with anthocyanin accumulation in cultured Pinot Noir grape berries.

Fruit quality is dependent on various factors including flavour, texture and colour. These factors are determined by the ripening process, either climacteric or non-climacteric. In grape berry, which is non-climacteric, the process is signalled by a complex set of hormone changes. Abscisic acid (ABA) is one of the key hormones involved in ripening, while sugar availability also plays a significant role in certain ripening aspects such as anthocyanin production. To understand the relative influence of hormone and sugar signalling in situ can prove problematic due to the physiological and environmental (abiotic and biotic) factors at play in vineyards. Here we report on the use of in vitro detached berry culture to investigate the comparative significance of ABA and sugar in the regulation of Pinot noir berry anthocyanin production under controlled conditions. Using a factorial experimental design, pre-véraison berries were cultured on media with various concentrations of sucrose and ABA. After 15 days of in vitro culture, the berries were analysed for changes in metabolites, hormones and gene expression. Results illustrated a stimulatory effect of sucrose and ABA on enhancing berry colour and a corresponding increase in anthocyanins. Increased ABA concentration was able to boost anthocyanin production in berries when sucrose supply was low. The sucrose and ABA effects on berry anthocyanins were primarily manifested through the up-regulation of transcription factors and other genes in the phenylpropanoid pathway, while in other parts of the pathway a down-regulation of key proanthocyanindin transcription factors and genes corresponded to sharp reduction in berry proanthocyanidins, irrespective of sucrose supply. Similarly, increased ABA was correlated with a significant reduction in berry malic acid and associated regulatory genes. These findings suggest a predominance of berry ABA over berry sugar in coordinating the physiological and genetic regulation of anthocyanins and proanthocyanins in Pinot noir grape berries.

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.

Elevating Ascorbate in Arabidopsis Stimulates the Production of Abscisic Acid, Phaseic Acid, and to a Lesser Extent Auxin (IAA) and Jasmonates, Resulting in Increased Expression of DHAR1 and Multiple Transcription Factors Associated with Abiotic Stress Tolerance.

Gene expression and phytohormone contents were measured in response to elevating ascorbate in the absence of other confounding stimuli such as high light and abiotic stresses. Young Arabidopsis plants were treated with 25 mM solutions of l-galactose pathway intermediates l-galactose (l-gal) or l-galactono-1,4-lactone (l-galL), as well as L-ascorbic acid (AsA), with 25 mM glucose used as control. Feeding increased rosette AsA 2- to 4-fold but there was little change in AsA biosynthetic gene transcripts. Of the ascorbate recycling genes, only Dehydroascorbate reductase 1 expression was increased. Some known regulatory genes displayed increased expression and included ANAC019, ANAC072, ATHB12, ZAT10 and ZAT12. Investigation of the ANAC019/ANAC072/ATHB12 gene regulatory network revealed a high proportion of ABA regulated genes. Measurement of a subset of jasmonate, ABA, auxin (IAA) and salicylic acid compounds revealed consistent increases in ABA (up to 4.2-fold) and phaseic acid (PA; up to 5-fold), and less consistently certain jasmonates, IAA, but no change in salicylic acid levels. Increased ABA is likely due to increased transcripts for the ABA biosynthetic gene NCED3. There were also smaller increases in transcripts for transcription factors ATHB7, ERD1, and ABF3. These results provide insights into how increasing AsA content can mediate increased abiotic stress tolerance.

Modifying Carbohydrate Supply to Fruit during Development Changes the Composition and Flavour of Actinidia chinensis var. chinensis 'Zesy002' Kiwifruit.

Consumer acceptance of fruit is determined by size, flavour and ripeness. In this study we investigated how altering the carbohydrate supply to Actinidia chinensis var. chinensis 'Zesy002' kiwifruit altered the balance between growth and accumulation of metabolites. Canes were phloem girdled and fruit thinned to a leaf-to-fruit ratio (L:F) of either 2 (Low carbohydrate) or 6 (High carbohydrate) at either 38 (Early) or 86 (Late) days after anthesis (DAA) and compared with ungirdled control canes with a L:F of 3. Fruit growth, metabolite accumulation, cytokinin concentrations and maturation were monitored and the sensory attributes of ripe fruit were assessed. The final weight of Early-High and Late-High carbohydrate fruit was 38% and 16% greater compared with control fruit. High carbohydrate fruit had increased starch, soluble sugar and cytokinin concentrations and fruit began to mature earlier and those with a Low carbohydrate had decreased concentrations and matured later compared with control fruit. Control fruit were described by consumers as more acidic and under-ripe compared with those from Early-High carbohydrate canes, but as sweeter than those from Low carbohydrate canes. This study showed that carbohydrate supply can have a major impact on the growth, sugar accumulation and maturity of 'Zesy002' fruit sinks.

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.

Boysenberry and apple juice concentrate reduced acute lung inflammation and increased M2 macrophage-associated cytokines in an acute mouse model of allergic airways disease.

Bioactive compounds including anthocyanins and other polyphenols are associated with reduced lung inflammation and improved lung function in asthma and other lung diseases. This study investigated the effects of a Boysenberry and apple juice concentrate, high in cyanidin glycosides, ellagitannins, and chlorogenic acid, on a mouse model of allergic airways inflammation. Male C57BL/6J mice were orally gavaged with 2.5 mg/kg of total anthocyanins (TAC) from BerriQi® Boysenberry and apple juice concentrate (0.2 mg/kg human equivalent dose) or water control 1 hr before an acute intranasal ovalbumin (OVA) challenge and were gavaged again 2 days after the intranasal challenge. Consumption of BerriQi® Boysenberry and apple juice concentrate significantly decreased OVA-induced infiltrating eosinophils, neutrophils, and T cells in the lung, and mucous production. Quantification of gene expression for arginase (Arg1), chitinase 3-like 3 (Ym-1), found in inflammatory zone (Fizz1), which have been associated with an anti-inflammatory macrophage phenotype (M2), found significantly increased Arg1 expression in the lung in the Boysenberry and apple juice concentrate treatment group. There was also increased production of M2-associated cytokines C-X-C motif chemokine ligand (CXCL) 10 and C-C motif chemokine ligand (CCL) 4. These results suggest that consumption of BerriQi® Boysenberry and apple juice concentrate promoted a shift toward an anti-inflammatory environment within the lung leading to reduced immune cell infiltration and tissue damage.

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.

Shy Girl, a kiwifruit suppressor of feminization, restricts gynoecium development via regulation of cytokinin metabolism and signalling.

Kiwifruit (Actinidia chinensis) is a dioecious, long-living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate alternative farming for better food security in the face of climate change. Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male-specific Shy Girl (SyGl) gene with homology to type-C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis 'Bruce'. Biallelic mutations of CEN and CEN4 generated rapid-flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid-flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self-pollinate and produce fast-flowering offspring. These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.

The effects of blanching on composition and modification of proteins in navy beans (Phaseolus vulgaris).

Blanching is an important process in the preparation of navy beans (Phaseolus vulgaris L.) for canning. We here explore the effect of blanching which can profoundly affect protein composition and introduce protein-primary-level modifications. Amino acid analysis showed significantly decreased protein abundance (58.5%) in blanched beans compared to raw beans. Proteomic analyses revealed a decrease in high molecular weight isoforms of the major storage globulin proteins phaseolin (mean fold-change -3.7) and legumin (mean fold-change -2.5) and concomitant increase in their low molecular weight isoforms (mean fold-change 6.4 and 8.3, respectively). Blanched beans also had decreased abundance of lipoxygenase (mean fold-change -13.1), an enzyme responsible for product spoilage during storage. Increased lysinoalanine (up to 47%) and highly modified protein fragments were found in the processing waters, indicating heat- induced modifications. Correlating these molecular level changes thus provides a basis for evaluating how processing parameters can be modified to increase protein food quality.

Defence Responses Associated with Elicitor-Induced, Cultivar-Associated Resistance to Latania Scale in Kiwifruit.

Latania scale insect is a pest of global significance affecting kiwifruit. The sessile insect (life stage: settled crawler-mature adult) is covered with a waxy cap that protects it from topical pesticides, so increasingly, a selection of resistant cultivars and application of elicitors are being used in pest control. Thus far, the application of a salicylic acid (SA) phytohormone pathway elicitor, acibenzolar-S-methyl (ASM), has been shown to reduce insect development (as indicated by cap size) on one kiwifruit cultivar ('Hayward'). To investigate how cultivar-associated resistance is affected by the ability to respond to different elicitors, we measured phytohormones (by LCMS) and gene expression (by qPCR and NanoString) on latania scale-tolerant 'Hort16A' and susceptible 'Hayward' kiwifruit over two seasons. Potted plants in the presence/absence of settled latania scales were treated with ASM (0.2 g/L) or methyl jasmonate (MeJA, 0.05% v/v), representing elicitors of the SA and JA signalling pathways, respectively. 'Hort16A' cultivar resistance to latania scale was associated with elevated expression of SA and SA-related defence genes (PR1 and two PR2 family genes) in the ASM treatment. MeJA treatments did not significantly affect insect development in 'Hayward' (latania scale did not survive on 'Hort16A') and did not correlate with phytohormone and gene expression measurements in either cultivar. 'Hayward' had greater concentrations than 'Hort16A' of inert storage forms of both SA and JA across all treatments. This information contributes to the selection of tolerant cultivars and the effective use of elicitors for control of latania scale in kiwifruit.

Cohort Profile: The Christchurch IBS cOhort to investigate Mechanisms FOr gut Relief and improved Transit (COMFORT).

BACKGROUND AND AIMS: This cross-sectional observational case-control study was initiated in July 2016 with the aim of increasing an understanding of the underlying disease mechanisms in functional gastrointestinal disorders (FGIDs) including irritable bowel syndrome (IBS), functional diarrhoea (FD), and functional constipation (FC). Specific areas of interest include the effect of food, microbiome, host and microbial genetics, metabolome, and psychological variables on unexplained chronic gastrointestinal (GI) symptoms. METHODS: This study recruited consecutive patients who were attending one of two endoscopy centres in Christchurch, New Zealand, for colonoscopy and a subgroup of participants from the general public who did not undergo colonoscopy. Participants with known GI disease other than an FGID were excluded. Those with symptoms were recruited as cases, whilst those without symptoms were recruited as controls. In the days prior to preparation for colonoscopy, or an agreeable time for those not undergoing colonoscopy, demographic, symptom, psychological, dietary, and health data were collected in addition to biological samples (breath, faeces, blood, and urine). Colonic biopsies were taken at the time of colonoscopy from participants in the colonoscopy subgroup. RESULTS: Between July 2016 and December 2018, 349 participants were recruited, 315 of whom completed the study, 220 participants were from the colonoscopy subgroup, and 95 from the non-colonoscopy subgroup. This included 129 controls and 186 cases (57 IBS-diarrhoea predominant, 30 IBS-constipation predominant, 41 IBS-mixed, 42 FC, and 16 FD). The mean age of FGID cases was 53.4 years and controls 54.4 years. Cases (149/186, 80.1%) and controls (57/72, 55.8%) were predominantly female. Education levels were similar across the cohort. Smoking and alcohol rates were also similar. Biological samples were collected as planned from participants. CONCLUSIONS: The COMFORT cohort is a unique clinical cohort of FGID cases and controls with a wide range of demographic, dietary, clinical, psychological, and health data in addition to biological samples. Future research will aim to use a systems biology approach to establish the potential role of diet, host-microbiome interactions, and other factors in the pathogenesis of FGIDs.

Bioaminergic Responses in an In Vitro System Studying Human Gut Microbiota-Kiwifruit Interactions.

Whole kiwifruit ('Hayward' and 'Zesy002') were examined for their bioaminergic potential after being subjected to in vitro gastrointestinal digestion and colonic fermentation. Controls included the prebiotic inulin and water, a carbohydrate-free vehicle. The dopamine precursor l-dihydroxyphenylalanine (L-DOPA) and the serotonin precursor 5-hydroxytryptophan were increased in the kiwifruit gastrointestinal digesta ('Hayward' > 'Zesy002') in comparison to the water digesta. Fermentation of the digesta with human fecal bacteria for 18 h modulated the concentrations of bioamine metabolites. The most notable were the significant increases in L-DOPA ('Zesy002' > 'Hayward') and γ-aminobutyric acid (GABA) ('Hayward' > 'Zesy002'). Kiwifruit increased Bifidobacterium spp. and Veillonellaceae (correlating with L-DOPA increase), and Lachnospira spp. (correlating with GABA). The digesta and fermenta were incubated with Caco-2 cells for 3 h followed by gene expression analysis. Effects were seen on genes related to serotonin synthesis/re-uptake/conversion to melatonin, gut tight junction, inflammation and circadian rhythm with different digesta and fermenta from the four treatments. These indicate potential effects of the substrates and the microbially generated organic acid and bioamine metabolites on intestinal functions that have physiological relevance. Further studies are required to confirm the potential bioaminergic effects of gut microbiota-kiwifruit interactions.

The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway.

The groups of plant flavonoid metabolites termed anthocyanins and proanthocyanins (PA) are responsible for pigmentation in seeds, flowers and fruits. Anthocyanins and PAs are produced by a pathway of enzymes which are transcriptionally regulated by transcription factors (TFs) that form the MYB-bHLH-WD40 (MBW) complex. In this study, transcriptomic analysis of purple-pigmented kiwifruit skin and flesh tissues identified MYBC1, from subgroup 5 of the R2R3 MYB family, and WRKY44 (highly similar to Arabidopsis TTG2) as candidate activators of the anthocyanin pathway. Transient over-expression of MYBC1 and WRKY44 induced anthocyanin accumulation in tobacco leaves. Dual luciferase promoter activation assays revealed that both MYBC1 and WRKY44 were able to strongly activate the promoters of the kiwifruit F3'H and F3'5'H genes. These enzymes are branch points of the pathway which specifies the type of anthocyanin accumulated. Stable over-expression of MYBC1 and WRKY44 in kiwifruit calli activated the expression of F3'5'H and PA-related biosynthetic genes as well as increasing levels of PAs. These results suggest that while previously characterised anthocyanin activator MYBs regulate the overall anthocyanin biosynthesis pathway, the PA-related TFs, MYBC1 and WRKY44, more specifically regulate key branch points. This adds a layer of regulatory control that potentially balances anthocyanin and PA levels.

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.

Phytohormone and Transcriptomic Analysis Reveals Endogenous Cytokinins Affect Kiwifruit Growth under Restricted Carbon Supply.

Following cell division, fruit growth is characterized by both expansion through increases in cell volume and biomass accumulation in cells. Fruit growth is limited by carbon starvation; however, the mechanism controlling fruit growth under restricted carbohydrate supply is poorly understood. In a previous study using red-fleshed kiwifruit, we showed that long-term carbon starvation had detrimental effects on carbohydrate, anthocyanin metabolism, and fruit growth. To elucidate the mechanisms underlying the reduction in fruit growth during kiwifruit development, we integrated phytohormone profiling with transcriptomic and developmental datasets for fruit under high or low carbohydrate supplies. Phytohormone profiling of the outer pericarp tissue of kiwifruit showed a 6-fold reduction in total cytokinin concentrations in carbon-starved fruit, whilst other hormones were less affected. Principal component analysis visualised that cytokinin composition was distinct between fruit at 16 weeks after mid bloom, based on their carbohydrate supply status. Cytokinin biosynthetic genes (IPT, CYP735A) were significantly downregulated under carbon starvation, in agreement with the metabolite data. Several genes that code for expansins, proteins involved in cell wall loosening, were also downregulated under carbon starvation. In contrast to other fleshy fruits, our results suggest that cytokinins not only promote cell division, but also drive fruit cell expansion and growth in kiwifruit.