A metabolomics-based approach to study abiotic stress in Lolium perenne

In the United Kingdom and Ireland, a major percentage of fertilized agricultural area is devoted to grasslands, which helps to support the associated milk and beef production industries. In temperate grasslands, perennial ryegrass (L. perenne) is the major forage grass and this species is particularly suitable as a forage grass due to its high yield and digestibility, when compared with other species. However, perennial ryegrass is not well adapted to abiotic stress conditions which are likely to occur in its natural environment. Some of the abiotic stress factors which have significant impacts on plant growth and development include water and nutrient availability. Therefore, this project set out to unravel some of the mechanisms involved in the adaptation of perennial ryegrass to limited water, phosphorous and nitrogen. In order to understand the metabolic mechanisms acting in response to these stresses, metabolite profiling was performed using GC-MS. Furthermore, for the water- and phosphorous-limitation studies this approach was complemented with transcript analysis.In order to study water-limitation a hydroponics system supplemented with polyethyleneglycol (PEG) was used to induce water-limitation for a period of one-week. A clear difference in the metabolic profiles of the leaves of plants grown under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more water stress-susceptible genotype Cashel and an increase in sugars and compatible solutes in the drought-tolerant PI 462336 genotype. Sugars exhibiting a significant increase included, raffinose, trehalose, glucose, fructose and maltose. Raffinose was identified as the metabolite exhibiting the largest accumulation under water-stress in the more tolerant genotype and may represent a target for engineering superior drought tolerance or form the basis of marker-assisted breeding in perennial ryegrass. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336 which identified genes in perennial ryegrass that were regulated by this stress.The characterization of the response to phosphorus-limitation was performed in a hydroponics system containing two solutions with different levels of phosphorus. Samples were collected from the roots and leaves of two genotypes 24 hours after being exposed to stress. Internal phosphate concentrations were reduced and significant alterations were detected in the metabolome and transcriptome of two perennial ryegrass genotypes. Results indicated a replacement of phospholipids with sulfolipids in response to P deficiency and that this occurs at the very early stages of P deficiency in perennial ryegrass. Additionally, the results suggested the role of glycolytic bypasses and the re-allocation of carbohydrates in response to P deficiency The characterization of the metabolic response of L. perenne leaves to different levels of nitrogen supply was performed for seven different genotypes with variability in the regrowth response rate to nitrogen supply in a hydroponics system. This facilitated the identification of common mechanisms of response between genotypes to nitrogen. The metabolic response observed included modifications of the lipid metabolism, as well as alterations of secondary aromatic metabolite precursors in plants exposed to nitrogendeficit. In contrast, plants grown in a nitrogen saturated media appeared to modify to some extent the metabolism of ascorbate. Additionally, it was found that amino acid levels increased with increasing concentrations of nitrogen supplied. This study suggested that the involvement of secondary metabolism, together with lipid and ascorbate metabolism, is of crucial importance in the early-adaptation of perennial ryegrass plants to different levels of nitrogen supply.EThOS - Electronic Theses Online ServiceIrish Department of Agriculture, Fisheries and Food (DAFF) (RSF 06 346)GBUnited Kingdo

A metabolomics-based approach to study abiotic stress in Lolium perenne

In the United Kingdom and Ireland, a major percentage of fertilized agricultural area is devoted to grasslands, which helps to support the associated milk and beef production industries. In temperate grasslands, perennial ryegrass (L. perenne) is the major forage grass and this species is particularly suitable as a forage grass due to its high yield and digestibility, when compared with other species. However, perennial ryegrass is not well adapted to abiotic stress conditions which are likely to occur in its natural environment. Some of the abiotic stress factors which have significant impacts on plant growth and development include water and nutrient availability. Therefore, this project set out to unravel some of the mechanisms involved in the adaptation of perennial ryegrass to limited water, phosphorous and nitrogen. In order to understand the metabolic mechanisms acting in response to these stresses, metabolite profiling was performed using GC-MS. Furthermore, for the water- and phosphorous-limitation studies this approach was complemented with transcript analysis.In order to study water-limitation a hydroponics system supplemented with polyethyleneglycol (PEG) was used to induce water-limitation for a period of one-week. A clear difference in the metabolic profiles of the leaves of plants grown under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more water stress-susceptible genotype Cashel and an increase in sugars and compatible solutes in the drought-tolerant PI 462336 genotype. Sugars exhibiting a significant increase included, raffinose, trehalose, glucose, fructose and maltose. Raffinose was identified as the metabolite exhibiting the largest accumulation under water-stress in the more tolerant genotype and may represent a target for engineering superior drought tolerance or form the basis of marker-assisted breeding in perennial ryegrass. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336 which identified genes in perennial ryegrass that were regulated by this stress.The characterization of the response to phosphorus-limitation was performed in a hydroponics system containing two solutions with different levels of phosphorus. Samples were collected from the roots and leaves of two genotypes 24 hours after being exposed to stress. Internal phosphate concentrations were reduced and significant alterations were detected in the metabolome and transcriptome of two perennial ryegrass genotypes. Results indicated a replacement of phospholipids with sulfolipids in response to P deficiency and that this occurs at the very early stages of P deficiency in perennial ryegrass. Additionally, the results suggested the role of glycolytic bypasses and the re-allocation of carbohydrates in response to P deficiency The characterization of the metabolic response of L. perenne leaves to different levels of nitrogen supply was performed for seven different genotypes with variability in the regrowth response rate to nitrogen supply in a hydroponics system. This facilitated the identification of common mechanisms of response between genotypes to nitrogen. The metabolic response observed included modifications of the lipid metabolism, as well as alterations of secondary aromatic metabolite precursors in plants exposed to nitrogendeficit. In contrast, plants grown in a nitrogen saturated media appeared to modify to some extent the metabolism of ascorbate. Additionally, it was found that amino acid levels increased with increasing concentrations of nitrogen supplied. This study suggested that the involvement of secondary metabolism, together with lipid and ascorbate metabolism, is of crucial importance in the early-adaptation of perennial ryegrass plants to different levels of nitrogen supply.EThOS - Electronic Theses Online ServiceIrish Department of Agriculture, Fisheries and Food (DAFF) (RSF 06 346)GBUnited Kingdo

Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites

peer-reviewedBackground Recent advances in the mapping of biochemical traits have been reported in Lolium perenne. Although the mapped traits, including individual sugars and fatty acids, contribute greatly towards ruminant productivity, organic acids and amino acids have been largely understudied despite their influence on the ruminal microbiome. Results In this study, we used a targeted gas-chromatography mass spectrometry (GC-MS) approach to profile the levels of 25 polar metabolites from different classes (sugars, amino acids, phenolic acids, organic acids and other nitrogen-containing compounds) present in a L. perenne F2 population consisting of 325 individuals. A quantitative trait (QTL) mapping approach was applied and successfully identified QTLs regulating seven of those polar metabolites (L-serine, L-leucine, glucose, fructose, myo-inositol, citric acid and 2, 3-hydroxypropanoic acid).Two QTL mapping approaches were carried out using SNP markers on about half of the population only and an imputation approach using SNP and DArT markers on the entire population. The imputation approach confirmed the four QTLs found in the SNP-only analysis and identified a further seven QTLs. Conclusions These results highlight the potential of utilising molecular assisted breeding in perennial ryegrass to modulate a range of biochemical quality traits with downstream effects in livestock productivity and ruminal digestion.This study was financed through a Research Stimulus Fund Grant by the Irish Department of Agriculture, Fisheries and Marine (RSF 06–346). AF, CH and DS acknowledge support from The Scottish Government’s Rural and Environment Science and Analytical Services Division

Bioprospection of Natural Sources of Polyphenols with Therapeutic Potential for Redox-Related Diseases

Funding: iNOVA4Health-UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is acknowledged. This work was supported by Fundação para a Ciência e Tecnologia (IF/01097/2013 to C.N.S.), by The Scottish Government Rural and Environment Science and Analytical Services Division (A.F. and D.S.), and BacHBerry FP7-KBBE-2013-613793 (R.M., A.F., C.J., I.C., G.G., R.R.-R., J.P., A.M., C.D., D.S. and C.N.S.). T.F.O. was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), and is currently supported by the DFG under Germany’s Excellence Strategy—EXC 2067/1-390729940.Plants are a reservoir of high-value molecules with underexplored biomedical applications. With the aim of identifying novel health-promoting attributes in underexplored natural sources, we scrutinized the diversity of (poly)phenols present within the berries of selected germplasm from cultivated, wild, and underutilized Rubus species. Our strategy combined the application of metabolomics, statistical analysis, and evaluation of (poly)phenols' bioactivity using a yeast-based discovery platform. We identified species as sources of (poly)phenols interfering with pathological processes associated with redox-related diseases, particularly, amyotrophic lateral sclerosis, cancer, and inflammation. In silico prediction of putative bioactives suggested cyanidin-hexoside as an anti-inflammatory molecule which was validated in yeast and mammalian cells. Moreover, cellular assays revealed that the cyanidin moiety was responsible for the anti-inflammatory properties of cyanidin-hexoside. Our findings unveiled novel (poly)phenolic bioactivities and illustrated the power of our integrative approach for the identification of dietary (poly)phenols with potential biomedical applications.publishersversionpublishe

Berry-Enriched Diet in Salt-Sensitive Hypertensive Rats:Metabolic Fate of (Poly)Phenols and the Role of Gut Microbiota

Diets rich in (poly)phenols are associated with a reduced reduction in the incidence of cardiovascular disorders. While the absorption and metabolism of (poly)phenols has been described, it is not clear how their metabolic fate is affected under pathological conditions. This study evaluated the metabolic fate of berry (poly)phenols in an in vivo model of hypertension as well as the associated microbiota response. Dahl salt-sensitive rats were fed either a low-salt diet (0.26% NaCl) or a high-salt diet (8% NaCl), with or without a berry mixture (blueberries, blackberries, raspberries, Portuguese crowberry and strawberry tree fruit) for 9 weeks. The salt-enriched diet promoted an increase in the urinary excretion of berry (poly)phenol metabolites, while the abundance of these metabolites decreased in faeces, as revealed by UPLC–MS/MS. Moreover, salt and berries modulated gut microbiota composition as demonstrated by 16S rRNA analysis. Some changes in the microbiota composition were associated with the high-salt diet and revealed an expansion of the families Proteobacteria and Erysipelotrichaceae. However, this effect was mitigated by the dietary supplementation with berries. Alterations in the metabolic fate of (poly)phenols occur in parallel with the modulation of gut microbiota in hypertensive rats. Thus, beneficial effects of (poly)phenols could be related with these interlinked modifications, between metabolites and microbiota environments.C.B., C.N.d.S., C.O. were funded by ANR (ANR-13-ISV1-0001-01) and FCT (FCTANR/BEX-BCM/0001/2013). D.B. was funded by the Austrian Science Fund (FWF P26127-B20) and European Research Council (Starting Grant: FunKeyGut 741623). D.S. and A.F. acknowledge support from Scottish Government: Rural and Environment Science and Analytical Services. We also acknowledge the Investment for the Future program ANR-11-IDEX-0003-01 within the LABEX ANR-10-LABX-0033 (C.B., C.O.), Fundação para a Ciência e Tecnologia financial support of A.G. (SFRH/BD/103155/2014) and C.N.d.S. (IF/01097/2013). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofounded by FCT through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged

RNA-seq, de novo transcriptome assembly and flavonoid gene analysis in 13 wild and cultivated berry fruit species with high content of phenolics

This research was funded by the European Union Framework Program 7, Project BacHBerry [FP7–613793]. The authors also acknowledge support from the Institute Strategic Programmes ‘Designing Future Wheat’ (BB/P016855/1), ‘Understanding and Exploiting Plant and Microbial Secondary Metabolism’ (BB/J004596/1) and ‘Molecules from Nature’ (BB/P012523/1) from the UK Biotechnology and Biological Sciences Research Council to the John Innes Centre and the European funded COST ACTION FA1106 QualityFruit. VT, PV and CM have also received funding from the European Union’s Horizon 2020 research and innovation programme through the TomGEM project under grant agreement No. 679796. The funding bodies had no role in the design of the study, collection, analysis and interpretation of data nor in writing the manuscript.Background: Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds. Results: To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige. Conclusions: We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.publishersversionpublishe

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project

Metabolomics: A High-throughput Screen for Biochemical and Bioactivity Diversity in Plants and Crops

Plants and crops contain a staggering diversity of compounds, many of which have pharmacological activity towards a variety of diseases. These properties have been exploited by traditional and modern medicine providing important sources of healthcare to this day. The contribution of natural products (such as plant-derived) to the modern pharmacopeia is indeed significant; however, the process of identifying novel bioactive compounds from biological sources has been a central challenge in the discovery of natural products. The resolution of these challenges relied extensively on the use of hyphenated Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR)-based analytical technologies for the structural elucidation and annotation of novel compounds. Technical developments in instrumentation and data processing have fostered the development of the field of metabolomics which provides a wealth of tools with the huge potential for application in the process of drug/bioactive discovery from plant tissues. This manuscript provides an overview of the metabolomics toolbox available for the discovery of novel bioactive compounds and the integration of these tools in the bioprospection and drug discovery workflows.Metabolomics: A High-throughput Screen for Biochemical and Bioactivity Diversity in Plants and CropsacceptedVersio