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Main conclusionGlycosylation from an anthocyanidin 3-O-glucosyltransferase Ps3GT (PsUGT78A27) facilitates the accumulation of pelargonidin 3-O-glucoside, which defines the vivid red flower color and occurs only in specific peony tree cultivars.Although tree peony cultivars of Chinese and Japanese both originated from China, vivid red color is only found in flowers of Japanese cultivars but not of Chinese cultivar groups. In this study, a Japanese tree peony cultivar ‘Taiyoh’ with vivid red petals and a Chinese tree peony cultivar ‘Hu Hong’ with reddish pink petals were chosen as the experimental materials. Flavonoids profiling indicated that pelargonidin 3-O-glucoside (Pg3G) detected only in Japanese cultivar contributed to vivid red color of tree peony petals, while pelargonidin 3,5-di-O-glucoside (Pg3G5G) found in both of Japanese and Chinese cultivars was responsible for pink flower color. Through the integration of full-length transcriptome sequencing and in vitro enzymatic activity analysis, two anthocyanin glucosyltransferase genes PsUGT78A27 and PsUGT75L45 were isolated from the petals of tree peony, and their encoding products exhibited enzymatic activities of pelargonidin 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively. Further quantitative real-time PCR revealed that PsUGT78A27 displayed high expression in petals of both cultivars and PsUGT75L45 was expressed at high levels in cultivar ‘Hu Hong’ only. Using a gene gun technique, the GFP fusion proteins of PsUGT78A27 and PsUGT75L45 were visualized to be cytoplasmic and nuclear localization in the epidermal cells of tree peony petals, and the glucosylation function of PsUGT78A27 and PsUGT75L45 to alter petal color of tree peony and herbaceous peony had been directly validated in vivo. These results demonstrated that PsUGT78A27 and PsUGT75L45 are key players for the presence or absence of vivid red flower color in tree peony cultivars. Our findings further elucidated the chemical and molecular mechanism of petal pigmentation of Paeonia and could help breed the Paeonia cultivars possessing novel flower colors.

Purpose To determine whether the anthocyanin, pelargonidin 3-glucoside (P3G), attenuates symptoms of inflammatory bowel disease (IBD) and metabolic syndrome in rats. Methods We tested P3G-enriched strawberry in two models of chronic inflammation in rats, chronic IBD induced by 0.5% dextran sodium sulphate in the drinking water for 12 weeks (D) and metabolic syndrome induced by a high-carbohydrate, high-fat diet (H) for 16 weeks. P3G-enriched strawberry was added to the diet for the final 6 weeks in IBD rats (DP) or 8 weeks in H rats (HP) to provide a dose of 8 mg P3G/kg/day. Results D rats had marked diarrhoea, bloody stools, erosion of mucosal epithelium, crypt atrophy, loss of villi and goblet cells, and inflammatory cell infiltration. These symptoms were reversed by P3G with healthy stools and mucosal lining of ileum and colon including increased villi, crypts and goblet cells and reduced inflammation. H rats developed hypertension, dyslipidaemia, central obesity, increased ventricular stiffness, cardiac and liver inflammation, and steatosis. P3G treatment in H rats improved systolic blood pressure, ventricular stiffness, and cardiac and liver structure, and reduced abdominal fat, abdominal circumference and body weight gain. Conclusions Our study indicates that dietary P3G decreased inflammation to decrease the symptoms of IBD, and to improve cardiovascular, liver and metabolic functions in metabolic syndrome.

Background Fragaria nilgerrensis (FN) provides a rich source of genetic variations for strawberry germplasm innovation. The color of strawberry fruits is a key factor affecting consumer preferences. However, the genetic basis of the fruit color formation in F. nilgerrensis and its interspecific hybrids has rarely been researched. Results In this study, the fruit transcriptomes and flavonoid contents of FN (white skin; control) and its interspecific hybrids BF1 and BF2 (pale red skin) were compared. A total of 31 flavonoids were identified. Notably, two pelargonidin derivatives (pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside) were revealed as potential key pigments for the coloration of BF1 and BF2 fruits. Additionally, dihydroflavonol 4-reductase ( DFR ) (LOC101293459 and LOC101293749) and anthocyanidin 3-O-glucosyltransferase ( BZ1 ) (LOC101300000), which are crucial structural genes in the anthocyanidin biosynthetic pathway, had significantly up-regulated expression levels in the two FN interspecific hybrids. Moreover, most of the genes encoding transcription factors (e.g., MYB, WRKY, TCP, bHLH, AP2, and WD40) related to anthocyanin accumulation were differentially expressed. We also identified two DFR genes (LOC101293749 and LOC101293459) that were significantly correlated with members in bHLH, MYB, WD40, AP2, and bZIP families. Two chalcone synthase ( CHS ) (LOC101298162 and LOC101298456) and a BZ1 gene (LOC101300000) were highly correlated with members in bHLH, WD40 and AP2 families. Conclusions Pelargonidin-3-O-glucoside and pelargonidin-3-O-rutinoside may be the key pigments contributing to the formation of pale red fruit skin. DFR and BZ1 structural genes and some bHLH, MYB, WD40, AP2, and bZIP TF family members enhance the accumulation of two pelargonidin derivatives. This study provides important insights into the regulation of anthocyanidin biosynthesis in FN and its interspecific hybrids. The presented data may be relevant for improving strawberry fruit coloration via genetic engineering.

Anthocyanins are a group of water-soluble pigments that confer the blue, purple, and red color to many fruits. Anthocyanin-rich fruits can be divided into three groups based on the types of aglycones of their anthocyanins: pelargonidin group, cyanidin/peonidin group, and multiple anthocyanidins group. Some fruits contain a major anthocyanin type and can serve as useful research tools. Cyanidin glycosides and peonidin glycosides can be metabolically converted to each other by methylation and demethylation. Both cyanidin and peonidin glycosides can be metabolized to protocatechuic acid and vanillic acid. Pelargonidin-3-glucoside is metabolized to 4-hydroxybenoic acid. On the other hand, phenolic acid metabolites of delphinidin, malvidin, and petunidin glycosides are unstable and can be further fragmented into smaller molecules. A literature review indicates berries with higher cyanidin content, such as black raspberries, chokeberries, and bilberries are more likely to produce an antiinflammatory effect. This observation seems to be consistent with the hypothesis that one or more stable phenolic acid metabolites contribute to the antiinflammatory effects of anthocyanin-rich fruits. More studies are needed before we can conclude that fruits rich in cyanidin, peonidin, or pelargonidin glycosides have better antiinflammatory effects. Additionally, fruit polyphenols other than anthocyanins could contribute to their antiinflammatory effects. Furthermore, blueberries could exert their health effects with other mechanisms such as improving intestinal microbiota composition. In summary, this classification system can facilitate our understanding of the absorption and metabolic processes of anthocyanins and the health effects of different fruits. •Fruits are divided into pelargonidin, cyanidin/peonidin, and multiple anthocyanins groups.•Cyanidin and peonidin glycosides can be metabolically converted to each other.•Cyanidin, peonidin, and pelargonidin glycosides can be metabolized to stable metabolites.•Higher cyanidin and pelargonidin content may be associated with antiinflammatory effect.•Antiinflammatory effect may be attributed to the stable phenolic acid metabolites.

Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies. IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts. To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.

Summary Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

Crop plant resistance against pathogens is governed by dynamic molecular and biochemical responses driven by complex transcriptional networks. However, the underlying mechanisms are largely unclear. Here we report an interesting role of HvWRKY23 transcription factor (TF) in modulating defense response against Fusarium head blight (FHB) in barley. The combined approach of gene silencing, metabolomics, real time expression analysis and ab initio bioinformatics tools led to the identification of the HvWRKY23 role in FHB resistance. The knock-down of HvWRKY23 gene in the FHB resistant barley genotype CI9831, followed by inoculation with Fusarium graminearum, led to the down regulation of key flavonoid and hydroxycinnamic acid amide biosynthetic genes resulting in reduced accumulation of resistant related (RR) secondary metabolites such as pelargonidin 3-rutinoside, peonidin 3-rhamnoside-5-glucoside, kaempferol 3-O-arabinoside and other flavonoid glycosides. Reduced abundances of RR metabolites in TF silenced plants were also associated with an increased proportion of spikelets diseased and amount of fungal biomass in spikelets, depicting the role of HvWRKY23 in disease resistance. The luciferase reporter assay demonstrated binding of HvWRKY23 protein to promoters of key flavonoid and hydroxycinnamic acid amides (HCAA) biosynthetic genes, such as HvPAL2, HvCHS1, HvHCT, HvLAC15 and HvUDPGT. The accumulation of high abundances of HCAAs and flavonoid glycosides reinforce cell walls to contain the pathogen to initial infection area. This gene in commercial cultivars can be edited, if non-functional, to enhance resistance against FHB.

Background Plants have remarkable diversity in petal colour through the biosynthesis and accumulation of various pigments. To better understand the mechanisms regulating petal pigmentation in Lonicera japonica , we used multiple approaches to investigate the changes in carotenoids, anthocyanins, endogenous hormones and gene expression dynamics during petal colour transitions, i.e., green bud petals (GB_Pe), white flower petals (WF_Pe) and yellow flower petals (YF_Pe). Results Metabolome analysis showed that YF_Pe contained a much higher content of carotenoids than GB_Pe and WF_Pe, with α-carotene, zeaxanthin, violaxanthin and γ-carotene identified as the major carotenoid compounds in YF_Pe. Comparative transcriptome analysis revealed that the key differentially expressed genes (DEGs) involved in carotenoid biosynthesis, such as phytoene synthase , phytoene desaturase and ζ-carotene desaturase , were significantly upregulated in YF_Pe. The results indicated that upregulated carotenoid concentrations and carotenoid biosynthesis-related genes predominantly promote colour transition. Meanwhile, two anthocyanins (pelargonidin and cyanidin) were significantly increased in YF_Pe, and the expression level of an anthocyanidin synthase gene was significantly upregulated, suggesting that anthocyanins may contribute to vivid yellow colour in YF_Pe. Furthermore, analyses of changes in indoleacetic acid, zeatin riboside, gibberellic acid, brassinosteroid (BR), methyl jasmonate and abscisic acid (ABA) levels indicated that colour transitions are regulated by endogenous hormones. The DEGs involved in the auxin, cytokinin, gibberellin, BR, jasmonic acid and ABA signalling pathways were enriched and associated with petal colour transitions. Conclusion Our results provide global insight into the pigment accumulation and the regulatory mechanisms underlying petal colour transitions during the flower development process in L. japonica .

Background: Strawberries have been identified to have antioxidant and anti-inflammatory properties that improve neuronal function and cognition, mostly in animal studies. It is unknown if the consumption of strawberries or related bioactives may reduce the risk of Alzheimer’s dementia risk. Material and Methods: The study was conducted in 925 participants, aged 58–98 years of the Rush Memory and Aging Project. Participants were dementia-free at baseline, completed a food frequency questionnaire, and had at least two annual neurological evaluations. The diagnosis of Alzheimer’s dementia was based on structured clinical neurological examination and standardized diagnostic criteria. The association of strawberry intake and incident Alzheimer’s dementia was analyzed using proportional hazard models adjusted for age, sex, education, physical activity, participation in cognitive activities, APOE-ɛ4 genotype, dietary intake of other fruits, and total calorie intake. Results: A total of 245 participants developed Alzheimer’s dementia over the mean follow-up of 6.7 (±3.6) years. Higher strawberry intake was associated with reduced risk of Alzheimer’s dementia (HR = 0.76, 95% CI: 0.60–0.96). In separate adjusted models, highest vs. lowest quartile intakes of Vitamin C (HR = 0.64, 95% CI: 0.45, 0.92), Pelargonidin (0.63, 95% CI: 0.43, 0.92), total anthocyanidins (0.69, 95% CI: 0.48, 0.99), and total flavonoids (0.67, 95% CI: 0.46, 0.98) were each associated with lower Alzheimer’s dementia risk. These associations remained after further adjustment for cardiovascular conditions. Conclusion: Consumption of strawberries and foods rich in vitamin C, pelargonidin, anthocyanidins, and total flavonoids may reduce the risk of Alzheimer’s dementia.

The CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.Key messageGenome edited poinsettia plants show loss-of-function mutations in the F3’H gene introduced by CRISPR/Cas9. This is sufficient to change the bract colour from red to reddish orange.