Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants.

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name "auronidin," with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

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.

UVR8-mediated induction of flavonoid biosynthesis for UVB tolerance is conserved between the liverwort Marchantia polymorpha and flowering plants.

Damaging UVB radiation is a major abiotic stress facing land plants. In angiosperms the UV RESISTANCE LOCUS8 (UVR8) photoreceptor coordinates UVB responses, including inducing biosynthesis of protective flavonoids. We characterised the UVB responses of Marchantia polymorpha (marchantia), the model species for the liverwort group of basal plants. Physiological, chemical and transcriptomic analyses were conducted on wild-type marchantia exposed to three different UVB regimes. CRISPR/Cas9 was used to obtain plant lines with mutations for components of the UVB signal pathway or the flavonoid biosynthetic pathway, and transgenics overexpressing the marchantia UVR8 sequence were generated. The mutant and transgenic lines were analysed for changes in flavonoid content, their response to UVB exposure, and transcript abundance of a set of 48 genes that included components of the UVB response pathway characterised for angiosperms. The marchantia UVB response included many components in common with Arabidopsis, including production of UVB-absorbing flavonoids, the central activator role of ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1). Notable differences included the greater importance of CHALCONE ISOMERASE-LIKE (CHIL). Mutants disrupted in the response pathway (hy5) or flavonoid production (chalcone isomerase, chil) were more easily damaged by UVB. Mutants (rup1) or transgenics (35S:MpMYB14) with increased flavonoid content had increased UVB tolerance. The results suggest that UVR8-mediated flavonoid induction is a UVB tolerance character conserved across land plants and may have been an early adaptation to life on land.

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.

Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants.

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization.

Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica).

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development.

Genetic, metabolite and developmental determinism of fruit friction discolouration in pear.

BACKGROUND: The unattractive appearance of the surface of pear fruit caused by the postharvest disorder friction discolouration (FD) is responsible for significant consumer dissatisfaction in markets, leading to lower returns to growers. Developing an understanding of the genetic control of FD is essential to enable the full application of genomics-informed breeding for the development of new pear cultivars. Biochemical constituents [phenolic compounds and ascorbic acid (AsA)], polyphenol oxidase (PPO) activity, as well as skin anatomy, have been proposed to play important roles in FD susceptibility in studies on a limited number of cultivars. However, to date there has been no investigation on the biochemical and genetic control of FD, employing segregating populations. In this study, we used 250 seedlings from two segregating populations (POP369 and POP356) derived from interspecific crosses between Asian (Pyrus pyrifolia Nakai and P. bretschneideri Rehd.) and European (P. communis) pears to identify genetic factors associated with susceptibility to FD. RESULTS: Single nucleotide polymorphism (SNP)-based linkage maps suitable for QTL analysis were developed for the parents of both populations. The maps for population POP369 comprised 174 and 265 SNP markers for the male and female parent, respectively, while POP356 maps comprised 353 and 398 SNP markers for the male and female parent, respectively. Phenotypic data for 22 variables were measured over two successive years (2011 and 2012) for POP369 and one year (2011) only for POP356. A total of 221 QTLs were identified that were linked to 22 phenotyped variables, including QTLs associated with FD for both populations that were stable over the successive years. In addition, clear evidence of the influence of developmental factors (fruit maturity) on FD and other variables was also recorded. CONCLUSIONS: The QTLs associated with fruit firmness, PPO activity, AsA concentration and concentration of polyphenol compounds as well as FD are the first reported for pear. We conclude that the postharvest disorder FD is controlled by multiple small effect QTLs and that it will be very challenging to apply marker-assisted selection based on these QTLs. However, genomic selection could be employed to select elite genotypes with lower or no susceptibility to FD early in the breeding cycle.

Dietary flavonoids from modified apple reduce inflammation markers and modulate gut microbiota in mice.

Apples are rich in polyphenols, which provide antioxidant properties, mediation of cellular processes such as inflammation, and modulation of gut microbiota. In this study we compared genetically engineered apples with increased flavonoids [myeloblastis transcription factor 10 (MYB10)] with nontransformed apples from the same genotype, "Royal Gala" (RG), and a control diet with no apple. Compared with the RG diet, the MYB10 diet contained elevated concentrations of the flavonoid subclasses anthocyanins, flavanol monomers (epicatechin) and oligomers (procyanidin B2), and flavonols (quercetin glycosides), but other plant secondary metabolites were largely unaltered. We used these apples to investigate the effects of dietary flavonoids on inflammation and gut microbiota in 2 mouse feeding trials. In trial 1, male mice were fed a control diet or diets supplemented with 20% MYB10 apple flesh and peel (MYB-FP) or RG apple flesh and peel (RG-FP) for 7 d. In trial 2, male mice were fed MYB-FP or RG-FP diets or diets supplemented with 20% MYB10 apple flesh or RG apple flesh for 7 or 21 d. In trial 1, the transcription levels of inflammation-linked genes in mice showed decreases of >2-fold for interleukin-2 receptor (Il2rb), chemokine receptor 2 (Ccr2), chemokine ligand 10 (Cxcl10), and chemokine receptor 10 (Ccr10) at 7 d for the MYB-FP diet compared with the RG-FP diet (P < 0.05). In trial 2, the inflammation marker prostaglandin E(2) (PGE(2)) in the plasma of mice fed the MYB-FP diet at 21 d was reduced by 10-fold (P < 0.01) compared with the RG-FP diet. In colonic microbiota, the number of total bacteria for mice fed the MYB-FP diet was 6% higher than for mice fed the control diet at 21 d (P = 0.01). In summary, high-flavonoid apple was associated with decreases in some inflammation markers and changes in gut microbiota when fed to healthy mice.

Triterpene acids from apple peel inhibit lepidopteran larval midgut lipases and larval growth.

Fruit extracts from apple, kiwifruit, feijoa, boysenberry, and blueberry were screened for the presence of lipase inhibitory compounds against lepidopteran larval midgut crude extracts. From 120 extracts, six showed significant inhibition with an extract from the peel of Malus × domestica cv. "Big Red" showing highest levels of inhibition. Because this sample was the only apple peel sample in the initial screen, a survey of peels from seven apple cultivars was undertaken and showed that, despite considerable variation, all had inhibitory activity. Successive solvent fractionation and LC-MS of cv. "Big Red" apple peel extract identified triterpene acids as the most important inhibitory compounds, of which ursolic acid and oleanolic acid were the major components and oxo- and hydroxyl-triterpene acids were minor components. When ursolic acid was incorporated into artificial diet and fed to Epiphyas postvittana Walker (Tortricidae: Lepidoptera) larvae at 0.16% w/v, a significant decrease in larval weight was observed after 21 days. This concentration of ursolic acid is less than half the concentration reported in the skin of some apple cultivars.

Apple polyphenol extracts protect against aspirin-induced gastric mucosal damage in rats.

The protective role of two apple polyphenol extracts, Douglas-FB (FB) and Douglas-EF (EF), on gastric mucosal damage following aspirin ingestion was investigated in healthy rats. Polyphenol content of the apple extracts varied, with the EF extract having 20% w/w polyphenols and a high proportion of flavanols as epicatechin and procyanidin, whereas the FB extract comprised 12% w/w polyphenols, which were mostly flavonols as quercetin glycosides. Male Sprague-Dawley rats were allocated to control, FB and EF groups and fed the experimental diet during the 10-day trial. Control treatment rats received 1 mL of deionised water, whereas apple polyphenol treatment group rats, FB and EF received a concentration of 10(-2) m polyphenols in 1 mL deionised water daily via oral gavage. At the end of 10-day feeding period, rats were fasted overnight, and the following morning, aspirin (200 mg/kg) was given by oral gavage. Four hours after aspirin administration, the animals were euthanised, and samples taken for analysis. Both apple polyphenol extracts significantly reduced the ulcer area, ulcer lesion index and gastric injury score. The glutathione in gastric mucosa was increased significantly in rats given FB apple extract. Despite their different polyphenol compositions, FB and EF apple extracts assisted in protecting the gastric mucosa following acute aspirin administration in rats.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population.

BACKGROUND: Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. RESULTS: Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals.A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia.The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. CONCLUSIONS: The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes.

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities.

PPARγ as a sensor of lipase activity and a target for the lipase inhibitor orlistat.

A PPARγ fluorescence polarization (FP) assay was used to measure the release of fatty acid products from triglyceride emulsions during digestion with pancreatic and yeast lipases in a real-time, homogenous assay. Using the same FP assay we show the anti-obesity drug Orlistat is a PPARγ ligand with an IC50 of 2.84 ± 0.16 µM. Analytical Mass Spectrometry confirms that Orlistat does not bind covalently to PPARγ. The PPARγ FP assay is shown to be a simple method for measuring real-time lipase activity using a number of triglyceride substrates including olive oil and grape seed oil emulsions. Incubation of Orlistat with the human intestinal epithelial cell line Caco-2, at concentrations of 1 - 100 µM, leads to induction of genes regulated by PPARγ. At 100 µM Orlistat, transcription of ß-defensin 1 (hDB1) & Adipose Differentiation Related Protein (ADRP) increase by up to 2.6 fold and 6.8 fold, respectively. Although at 1 µM and 100 µM Orlistat did not significantly increase defensin protein synthesis, at 10 µM Orlistat induced a 1.5 fold increase in hDB1 protein secretion in the human colonic adenocarcinoma cell line HT-29. Thus Orlistat is similar to the anti-diabetic drug Rosiglitazone in its ability to induce defensin gene expression. The antimicrobial peptide ß-defensin 1 protects against pathogenic micro-organisms in the gut and PPARγ suppresses inflammatory gene expression. These may be beneficial side effects of Orlistat consumption on gut epithelial cells.

Metabolite profiling and quantification of phytochemicals in potato extracts using ultra-high-performance liquid chromatography-mass spectrometry.

BACKGROUND: Potatoes contain a diverse range of phytochemicals which have been suggested to have health benefits. Metabolite profiling and quantification were conducted on plant extracts made from a white potato cultivar and 'Urenika', a purple potato cultivar traditionally consumed by New Zealand Maori. There is limited published information regarding the metabolite profile of Solanum tuberosum cultivar 'Urenika'. RESULTS: Using ultra-high- performance liquid chromatography-mass spectrometry (UHPLC-MS), a total of 31 compounds were identified and quantified in the potato extracts. The majority of the compounds were identified for the first time in 'Urenika'. These compounds include several types of anthocyanins, hydroxycinnamic acid (HCA) derivatives, and hydroxycinnamic amides (HCAA). Six classes of compounds, namely organic acids, amino acids, HCA, HCAA, flavonols and glycoalkaloids, were present in both extracts but quantities varied between the two extracts. CONCLUSIONS: The unknown plant metabolites in both potato extracts were assigned with molecular formulae and identified with high confidence. Quantification of the metabolites was achieved using a number of appropriate standards. High-resolution mass spectrometry data critical for accurate identification of unknown phytochemicals were achieved and could be added to potato or plant metabolomic database.

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.

QTL and candidate gene mapping for polyphenolic composition in apple fruit.

BACKGROUND: The polyphenolic products of the phenylpropanoid pathway, including proanthocyanidins, anthocyanins and flavonols, possess antioxidant properties that may provide health benefits. To investigate the genetic architecture of control of their biosynthesis in apple fruit, various polyphenolic compounds were quantified in progeny from a 'Royal Gala' × 'Braeburn' apple population segregating for antioxidant content, using ultra high performance liquid chromatography of extracts derived from fruit cortex and skin. RESULTS: Construction of genetic maps for 'Royal Gala' and 'Braeburn' enabled detection of 79 quantitative trait loci (QTL) for content of 17 fruit polyphenolic compounds. Seven QTL clusters were stable across two years of harvest and included QTLs for content of flavanols, flavonols, anthocyanins and hydroxycinnamic acids. Alignment of the parental genetic maps with the apple whole genome sequence in silico enabled screening for co-segregation with the QTLs of a range of candidate genes coding for enzymes in the polyphenolic biosynthetic pathway. This co-location was confirmed by genetic mapping of markers derived from the gene sequences. Leucoanthocyanidin reductase (LAR1) co-located with a QTL cluster for the fruit flavanols catechin, epicatechin, procyanidin dimer and five unknown procyanidin oligomers identified near the top of linkage group (LG) 16, while hydroxy cinnamate/quinate transferase (HCT/HQT) co-located with a QTL for chlorogenic acid concentration mapping near the bottom of LG 17. CONCLUSION: We conclude that LAR1 and HCT/HQT are likely to influence the concentration of these compounds in apple fruit and provide useful allele-specific markers for marker assisted selection of trees bearing fruit with healthy attributes.

Transcriptional analysis of apple fruit proanthocyanidin biosynthesis.

Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple.

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.