Tony K. McGhie

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a–c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85–95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a–c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g., in biofermentation and biopharming.

SUMMARY: Plant adaptation from aquatic to terrestrial environments required modifications to cell wall structure and function to provide tolerance to new abiotic and biotic stressors. Here, we investigate the nature and function of red auronidin pigment accumulation in the cell wall of the liverwort Marchantia polymorpha. Transgenic plants with auronidin production either constitutive or absent were analysed for their cell wall properties, including fractionation of polysaccharide and phenolic components. While small amounts of auronidin and other flavonoids were loosely associated with the cell wall, the majority of the pigments were tightly associated, similar to what is observed in angiosperms for polyphenolics such as lignin. No evidence of covalent binding to a polysaccharide component was found: we propose auronidin is present in the wall as a physically entrapped large molecular weight polymer. The results suggested auronidin is a dual function molecule that can both screen excess light and increase wall strength, hydrophobicity and resistance to enzymatic degradation by pathogens. Thus, liverworts have expanded the core phenylpropanoid toolkit that was present in the ancestor of all land plants, to deliver a lineage‐specific solution to some of the environmental stresses faced from a terrestrial lifestyle.

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.

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-β-glucoside” (AA-2βG), 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.

Apples are a commonly consumed fruit linked with reducing the risk of several chronic diseases, including cancer. Some of the health benefits of apples are related to their secondary metabolites, known as phytochemicals. The type of phytochemicals and their distribution differ within the various parts of an apple (skin vs. flesh) and are affected by cultivar type and food processing methods. Several studies have reported higher content of apple phytochemicals in the skin compared to the flesh, but the skin only makes up 10% of the weight of the apple. Only a few studies have reported on differences in the phytochemical content relative to the weight of the skin compared to the whole apple. In this comparative study, we used liquid chromatography–mass spectrometry (UPLC-QTOF-MSLC-MS) to identified phenolics (flavonoids and phenolic acids) and triterpenoids as the main phytochemicals in the skin and flesh of a New Zealand (NZ) heritage apple cultivar known as ‘Monty’s Surprise’. We further evaluate the effect of two simple household processing techniques, puree and air dehydration, on phytochemical concentration. Our preliminary analysis showed that ‘Monty’s Surprise’ contained a higher concentration of catechin, epicatechin, total procyanidins and chlorogenic acid when compared to the commercial apple cultivars ‘Red Delicious’ and ‘Braeburn’. Furthermore, flavonols (quercetin glycosides) were present mainly in the skin, whereas chlorogenic acid levels were higher in the flesh. Moreover, our data showed that apple puree retains a phytochemical concentration similar to that of fresh apple, whereas air dehydration appears to be more destructive to apple phytochemicals. The findings from this study point out that, to obtain the greatest health benefits from apples, it is important to consume the whole apple, including skin. Additionally, an apple’s health effects vary in relation to the phytochemical profile of the apple that is consumed. Furthermore, this study identifies a simple household processing method for long-term storage that preserves the apple’s phytochemical profile to achieve maximum health benefits.

ISHS X International Symposium on Kiwifruit Nutrient and phenolic composition of Zespri™ Red Kiwifruit

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.

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.

Summary: 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.

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.