Anthocyanins-gut microbiota-health axis: A review.

Anthocyanins are a subclass of flavonoids responsible for color in some fruits and vegetables with potent antioxidative capacity. During digestion, a larger proportion of dietary anthocyanins remains unabsorbed and reach the large intestine where they interact with the gut microbiota. Anthocyanins can modulate gut microbial populations to improve diversity and the proportion of beneficial populations, leading to alterations in short chain fatty acid and bile acid production. Some anthocyanins can be degraded into colonic metabolites, such as phenolic acids, which accumulate in the body and regulate a range of biological activities. Here we provide an overview of the effects of dietary anthocyanin consumption on gut microbial interactions, metabolism, and composition. Progression of chronic diseases has been strongly associated with imbalances in gut microbial populations. We therefore focus on the role of the gut microbiota as the 'mediator' that facilitates the therapeutic potential of anthocyanins against various chronic diseases, including obesity, type II diabetes, cardiovascular disease, neurodegenerative disease, inflammatory bowel disease, cancer, fatty liver disease, chronic kidney disease and osteoarthritis.

A Model to Incorporate the bHLH Transcription Factor OsIRO3 within the Rice Iron Homeostasis Regulatory Network.

Iron (Fe) homeostasis in plants is governed by a complex network of regulatory elements and transcription factors (TFs), as both Fe toxicity and deficiency negatively impact plant growth and physiology. The Fe homeostasis network is well characterized in Arabidopsis thaliana and remains poorly understood in monocotyledon species such as rice (Oryza sativa L.). Recent investigation of the rice Fe homeostasis network revealed OsIRO3, a basic Helix-Loop-Helix (bHLH) TF as a putative negative regulator of genes involved in Fe uptake, transport, and storage. We employed CRISPR-Cas9 gene editing to target the OsIRO3 coding sequence and generate two independent T-DNA-free, loss-of-function iro3 mutants in rice cv. Nipponbare. The iro3 mutant plants had similar phenotype under nutrient-sufficient conditions and had stunted growth under Fe-deficient conditions, relative to a T-DNA free, wild-type control (WT). Under Fe deficiency, iro3 mutant shoots had reduced expression of Fe chelator biosynthetic genes (OsNAS1, OsNAS2, and OsNAAT1) and upregulated expression of an Fe transporter gene (OsYSL15), relative to WT shoots. We place our results in the context of the existing literature and generate a model describing the role of OsIRO3 in rice Fe homeostasis and reinforce the essential function of OsIRO3 in the rice Fe deficiency response.

Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

Annotation and Molecular Characterisation of the TaIRO3 and TaHRZ Iron Homeostasis Genes in Bread Wheat (Triticum aestivum L.).

Effective maintenance of plant iron (Fe) homoeostasis relies on a network of transcription factors (TFs) that respond to environmental conditions and regulate Fe uptake, translocation, and storage. The iron-related transcription factor 3 (IRO3), as well as haemerythrin motif-containing really interesting new gene (RING) protein and zinc finger protein (HRZ), are major regulators of Fe homeostasis in diploid species like Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.), but remain uncharacterised in hexaploid bread wheat (Triticum aestivum L.). In this study, we have identified, annotated, and characterised three TaIRO3 homoeologs and six TaHRZ1 and TaHRZ2 homoeologs in the bread wheat genome. Protein analysis revealed that TaIRO3 and TaHRZ proteins contain functionally conserved domains for DNA-binding, dimerisation, Fe binding, or polyubiquitination, and phylogenetic analysis revealed clustering of TaIRO3 and TaHRZ proteins with other monocot IRO3 and HRZ proteins, respectively. Quantitative reverse-transcription PCR analysis revealed that all TaIRO3 and TaHRZ homoeologs have unique tissue expression profiles and are upregulated in shoot tissues in response to Fe deficiency. After 24 h of Fe deficiency, the expression of TaHRZ homoeologs was upregulated, while the expression of TaIRO3 homoeologs was unchanged, suggesting that TaHRZ functions upstream of TaIRO3 in the wheat Fe homeostasis TF network.

Effect of Rice GDP-L-Galactose Phosphorylase Constitutive Overexpression on Ascorbate Concentration, Stress Tolerance, and Iron Bioavailability in Rice.

Ascorbate (vitamin C) is an essential multifunctional molecule for both plants and mammals. In plants, ascorbate is the most abundant water-soluble antioxidant that supports stress tolerance. In humans, ascorbate is an essential micronutrient and promotes iron (Fe) absorption in the gut. Engineering crops with increased ascorbate levels have the potential to improve both crop stress tolerance and human health. Here, rice (Oryza sativa L.) plants were engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase coding sequence (35S-OsGGP), which encodes the rate-limiting enzymatic step of the L-galactose pathway. Ascorbate concentrations were negligible in both null segregant (NS) and 35S-OsGGP brown rice (BR, unpolished grain), but significantly increased in 35S-OsGGP germinated brown rice (GBR) relative to NS. Foliar ascorbate concentrations were significantly increased in 35S-OsGGP plants in the vegetative growth phase relative to NS, but significantly reduced at the reproductive growth phase and were associated with reduced OsGGP transcript levels. The 35S-OsGGP plants did not display altered salt tolerance at the vegetative growth phase despite having elevated ascorbate concentrations. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells - an accurate predictor of Fe bioavailability in human digestion - exposed to in vitro digests of NS and 35S-OsGGP BR and GBR samples.

Nicotianamine-chelated iron positively affects iron status, intestinal morphology and microbial populations in vivo (Gallus gallus).

Wheat flour iron (Fe) fortification is mandatory in 75 countries worldwide yet many Fe fortificants, such as Fe-ethylenediaminetetraacetate (EDTA), result in unwanted sensory properties and/or gastrointestinal dysfunction and dysbiosis. Nicotianamine (NA) is a natural chelator of Fe, zinc (Zn) and other metals in higher plants and NA-chelated Fe is highly bioavailable in vitro. In graminaceous plants NA serves as the biosynthetic precursor to 2' -deoxymugineic acid (DMA), a related Fe chelator and enhancer of Fe bioavailability, and increased NA/DMA biosynthesis has proved an effective Fe biofortification strategy in several cereal crops. Here we utilized the chicken (Gallus gallus) model to investigate impacts of NA-chelated Fe on Fe status and gastrointestinal health when delivered to chickens through intraamniotic administration (short-term exposure) or over a period of six weeks as part of a biofortified wheat diet containing increased NA, Fe, Zn and DMA (long-term exposure). Striking similarities in host Fe status, intestinal functionality and gut microbiome were observed between the short-term and long-term treatments, suggesting that the effects were largely if not entirely due to consumption of NA-chelated Fe. These results provide strong support for wheat with increased NA-chelated Fe as an effective biofortification strategy and uncover novel impacts of NA-chelated Fe on gastrointestinal health and functionality.

Genome-wide identification and characterization of the GDP-L-galactose phosphorylase gene family in bread wheat.

BACKGROUND: Ascorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting enzyme of the L-galactose pathway-the dominant ascorbate biosynthetic pathway in plants-and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5'-untranslated region (5'UTR). The GGP genes have yet to be identified in bread wheat (Triticum aestivum L.), one of the most important food grain sources for humans. RESULTS: Bread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes (TaGGP2-A/B/D and TaGGP1-A/B/D, respectively) and a highly conserved uORF was present in the long 5'UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta. CONCLUSIONS: We have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium/Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta. The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.

Investigation of Nicotianamine and 2' Deoxymugineic Acid as Enhancers of Iron Bioavailability in Caco-2 Cells.

Nicotianamine (NA) is a low-molecular weight metal chelator in plants with high affinity for ferrous iron (Fe2+) and other divalent metal cations. In graminaceous plant species, NA serves as the biosynthetic precursor to 2' deoxymugineic acid (DMA), a root-secreted mugineic acid family phytosiderophore that chelates ferric iron (Fe3+) in the rhizosphere for subsequent uptake by the plant. Previous studies have flagged NA and/or DMA as enhancers of Fe bioavailability in cereal grain although the extent of this promotion has not been quantified. In this study, we utilized the Caco-2 cell system to compare NA and DMA to two known enhancers of Fe bioavailability-epicatechin (Epi) and ascorbic acid (AsA)-and found that both NA and DMA are stronger enhancers of Fe bioavailability than Epi, and NA is a stronger enhancer of Fe bioavailability than AsA. Furthermore, NA reversed Fe uptake inhibition by Myricetin (Myr) more than Epi, highlighting NA as an important target for biofortification strategies aimed at improving Fe bioavailability in staple plant foods.

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability.

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up-regulate biosynthesis of two low molecular weight metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - that play key roles in metal transport and nutrition. The CE-OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X-ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field-grown CE-OsNAS2 grain and positively correlated with NA and DMA concentrations.

Investigation of Baseline Iron Levels in Australian Chickpea and Evaluation of a Transgenic Biofortification Approach.

Iron deficiency currently affects over two billion people worldwide despite significant advances in technology and society aimed at mitigating this global health problem. Biofortification of food staples with iron (Fe) represents a sustainable approach for alleviating human Fe deficiency in developing countries, however, biofortification efforts have focused extensively on cereal staples while pulses have been largely overlooked. In this study we describe a genetic engineering (GE) approach to biofortify the pulse crop, chickpea (Cicer arietinum L.), with Fe using a combination of the chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes which function in Fe transport and storage, respectively. This study consists of three main components: (1) the establishment for baseline Fe concentration of existing germplam, (2) the isolation and study of expression pattern of the novel CaNAS2 gene, and (3) the generation of GE chickpea overexpressing the CaNAS2 and GmFER genes. Seed of six commercial chickpea cultivars was collected from four different field locations in Australia and assessed for seed Fe concentration. The results revealed little difference between the cultivars assessed, and that chickpea seed Fe was negatively affected where soil Fe bioavailability is low. The desi cultivar HatTrick was then selected for further study. From it, the CaNAS2 gene was cloned and its expression in different tissues examined. The gene was found to be expressed in multiple vegetative tissues under Fe-sufficient conditions, suggesting that it may play a housekeeping role in systemic translocation of Fe. Two GE chickpea events were then generated and the overexpression of the CaNAS2 and GmFER transgenes confirmed. Analysis of nicotianamine (NA) and Fe levels in the GE seeds revealed that NA was nearly doubled compared to the null control while Fe concentration was not changed. Increased NA content in chickpea seed is likely to translate into increased Fe bioavailability and may thus overcome the effect of the bioavailability inhibitors found in pulses; however, further study is required to confirm this. This is the first known example of GE Fe biofortified chickpea; information gleaned from this study can feed into future pulse biofortification work to help alleviate global Fe deficiency.

Characterisation of the nicotianamine aminotransferase and deoxymugineic acid synthase genes essential to Strategy II iron uptake in bread wheat (Triticum aestivum L.).

Iron (Fe) uptake in graminaceous plant species occurs via the release and uptake of Fe-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the MAs biosynthetic pathway, nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) enzymes catalyse the formation of 2'-deoxymugineic acid (DMA) from nicotianamine (NA). Here we describe the identification and characterisation of six TaNAAT and three TaDMAS1 genes in bread wheat (Triticum aestivum L.). The coding sequences of all six TaNAAT homeologs consist of seven exons with ≥88.0% nucleotide sequence identity and most sequence variation present in the first exon. The coding sequences of the three TaDMAS1 homeologs consist of three exons with ≥97.8% nucleotide sequence identity. Phylogenetic analysis revealed that the TaNAAT and TaDMAS1 proteins are most closely related to the HvNAAT and HvDMAS1 proteins of barley and that there are two distinct groups of TaNAAT proteins-TaNAAT1 and TaNAAT2 -that correspond to the HvNAATA and HvNAATB proteins, respectively. Quantitative reverse transcription-PCR analysis revealed that the TaNAAT2 genes are expressed at highest levels in anther tissues whilst the TaNAAT1 and TaDMAS1 genes are expressed at highest levels in root tissues of bread wheat. Furthermore, the TaNAAT1, TaNAAT2 and TaDMAS1 genes were differentially regulated by plant Fe status and their expression was significantly upregulated in root tissues from day five onwards during a seven-day Fe deficiency treatment. The identification and characterization of the TaNAAT1, TaNAAT2 and TaDMAS1 genes provides a valuable genetic resource for improving bread wheat growth on Fe deficient soils and enhancing grain Fe nutrition.