Rapid UHPLC-MS metabolite profiling and phenotypic assays reveal genotypic impacts of nitrogen supplementation in oats

Abstract

Introduction Oats ( Avena sativa L.) are a whole grain cereal recognised for their health benefits and which are cultivated largely in temperate regions providing both a source of food for humans and animals, as well as being used in cosmetics and as a potential treatment for a number of diseases. Oats are known as being a cereal source high in dietary fibre (e.g. β-glucans), as well as being high in antioxidants, minerals and vitamins. Recently, oats have been gaining increased global attention due to their large number of beneficial health effects. Consumption of oats has been proven to lower blood LDL cholesterol levels and blood pressure, thus reducing the risk of heart disease, as well as reducing blood-sugar and insulin levels. Objectives Oats are seen as a low input cereal. Current agricultural guidelines on nitrogen application are believed to be suboptimal and only consider the effect of nitrogen on grain yield. It is important to understand the role of both variety and of crop management in determining nutritional quality of oats. In this study the response of yield, grain quality and grain metabolites to increasing nitrogen application to levels greater than current guidelines were investigated. Methods Four winter oat varieties (Mascani, Tardis, Balado and Gerald) were grown in a replicated nitrogen response trial consisting of a no added nitrogen control and four added nitrogen treatments between 50 and 200 kg N ha −1 in a randomised split-plot design. Grain yield, milling quality traits, β-glucan, total protein and oil content were assessed. The de-hulled oats (groats) were also subjected to a rapid Ultra High Performance Liquid Chromatography—Mass Spectrometry (UHPLC-MS) metabolomic screening approach. Results Application of nitrogen had a significant effect on grain yield but there was no significant difference between the response of the four varieties. Grain quality traits however displayed significant differences both between varieties and nitrogen application level. β-glucan content significantly increased with nitrogen application. The UHPLC-MS approach has provided a rapid, sub 15 min per sample, metabolite profiling method that is repeatable and appropriate for the screening of large numbers of cereal samples. The method captured a wide range of compounds, inclusive of primary metabolites such as the amino acids, organic acids, vitamins and lipids, as well as a number of key secondary metabolites, including the avenanthramides, caffeic acid, and sinapic acid and its derivatives and was able to identify distinct metabolic phenotypes for the varieties studied. Amino acid metabolism was massively upregulated by nitrogen supplementation as were total protein levels, whilst the levels of organic acids were decreased, likely due to them acting as a carbon skeleton source. Several TCA cycle intermediates were also impacted, potentially indic...

Assessing the impact of nitrogen supplementation in oats across multiple growth locations and years with targeted phenotyping and high-resolution metabolite profiling approaches

“…In addition, GC-MS provided a complementary approach better tailored to the retention of a wider range of amino acids and sugars (Table S4). Several updates to our previous oat UHPLC-PDA-MS method ( Allwood et al, 2019a ) have been made, including: 5.75-fold increase from 0.33 mg to 1.9 mg of groat material on-column; higher resolution column and high pressure capacity UHPLC system; and application of a longer gradient. As can be seen from the representative UHPLC chromatograms ( Fig.…”

“…The recently developed metabolite extract enrichment and UHPLC-PDA-MS/MS profiling methods have achieved greater levels of metabolite chromatographic resolution, as well as greater numbers of metabolites detected and reliably identified, in comparison to our previous rapid UHPLC-MS method ( Allwood et al, 2019a ). Both the GC-MS and UHPLC-MS profiling approaches, through applications of high levels of QA, have been shown to be highly repeatable and appropriate for the screening of large numbers of oat grain samples.…”

“…Grain numbers per m 2 and per panicle were calculated using grain yield and TGW results. Groats were obtained by passing 25 g of whole grain through a Laboratory Oat Huller (Codema Model LH5095; Maple Grove, Minneapolis, USA) set at 100 bar for 60 s. Groat nitrogen and oil content were predicted using near infrared spectroscopy (NIRS) and β-glucan content was determined with a Megazyme™ kit, both as described in Allwood et al (2019a) . These data were analysed using 2-way analysis of variance and Fisher’s protected least significance test using GENSTAT 16th edition and correlation analysis conducted using Microsoft Excel.…”

“…The de-hulled oats (groats) were also subjected to Gas Chromatography – Mass Spectrometry (GC-MS) analysis of polar metabolites. A newly developed extraction enrichment and high resolution Ultra High-Performance Liquid Chromatography (UHPLC)-MS/MS method, superseding a previous rapid (sub 15 min) method ( Allwood et al, 2019a ), was applied to profile a range of lipids, phenolic compounds, and a number of primary metabolites retained by C18 UHPLC.…”

Profiling and characterization of oat cultivars (Avena sativa L.) with respect to bioactive compounds, pesticide residues and mycotoxin

“…Contents were evaporated by using rotary evaporator (Büchi R-3000, Büchi Labortechnic AG, Postfach, Switzerland), and then was diluted with 1 mL of analytical grade acetone. [19] Organic sugars quantification Organic sugar quantification was carried out according to reported method of Allwood et al [20] HPLCgrade acetonitrile bought from Thermo Fisher Scientific (USA) and HPLC-grade methanol bought from Kermel Chemical Reagent Co. Ltd. (Tianjin, China) were utilized. In this study, deionized water was also used (Milli-Q, Millipore Corp., Milford, MA, USA) of 18.2 MΩ/cm resistivity.…”