Rapid UHPLC-MS metabolite profiling and phenotypic assays reveal genotypic impacts of nitrogen supplementation in oats
Rapid UHPLC-MS metabolite profiling and phenotypic assays reveal genotypic impacts of nitrogen supplementation in oats
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DOI：
10.1007/s11306-019-1501-x
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发表时间：
2019-03-01
期刊：
影响因子：
3.6
通讯作者：
Howarth, Catherine
中科院分区：
文献类型：
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作者：
Allwood, J. William;Xu, Yun;Howarth, Catherine
燕麦(Avena sativa L.)是一种被认为对健康有好处的全谷物谷物，主要在温带地区种植，为人类和动物提供食物来源，并被用于化妆品和一些疾病的潜在治疗。燕麦被认为是一种富含膳食纤维(如葡聚糖)的谷类食品，同时还富含抗氧化剂、矿物质和维生素。最近，燕麦因其对健康的大量有益作用而受到全球越来越多的关注。燕麦的消费已被证明可以降低血液中的低密度脂蛋白胆固醇水平和血压，从而降低心脏病的风险，并降低血糖和胰岛素水平。目前关于氮肥使用的农业指导方针被认为是次优的，只考虑了氮素对粮食产量的影响。重要的是要了解品种和作物管理在确定燕麦营养品质方面的作用。本研究以4个冬季燕麦品种(Mascani、Tardis、Balado和Gerald)为试材，采用随机裂区设计，在50~200kgNha(-1)范围内设置4个施氮量为50~200kgNha(-1)的施氮量对照和4个施氮量处理，研究了不同施氮量对产量、品质和籽粒代谢产物的影响。对籽粒产量、碾磨品质、-葡聚糖、总蛋白质和含油量进行了测定。同时对脱皮燕麦(GRAAT)进行了快速超高效液相色谱-质谱仪(UHPLC-MS)代谢体筛选。结果施氮量对籽粒产量有显著影响，但4个品种间的反应差异不显著。但籽粒品质性状在品种间和施氮量间均存在显着差异。-葡聚糖含量随施氮量的增加而显著增加。UHPLC-MS方法提供了一种快速的、每个样品不到15分钟的代谢物图谱分析方法，该方法可重复使用，适用于大量谷物样品的筛选。该方法捕获了广泛的化合物，包括氨基酸、有机酸、维生素和脂肪等初级代谢物，以及一些关键的次生代谢物，包括燕麦、咖啡酸和芥子酸及其衍生物，并能够识别所研究品种的不同代谢表型。补充氮素显著上调了氨基酸代谢和总蛋白质水平，同时降低了有机酸水平，这可能是因为有机酸是碳骨架来源。几个TCA循环中间体也受到了影响，这可能表明TCA循环周转率增加，从而为植物提供能量来源和还原能力，以帮助提高氮素同化。氮素供应的增加也有助于增加含氮磷脂的产量。结论尽管该方法作为一种快速筛选方法或快速代谢物图谱方法具有广泛的适用性，并在本研究中提供了有价值的代谢见解，但仍必须认为，应用高分辨率的层析方法将获得更大的代谢物鉴定的置信度和定量精密度，尽管以较大的样品吞吐量为代价。后续研究将应用更高分辨率的GC(气相色谱)和LC(反相和HILIC)方法，还将从多个生长地点和生长季节对燕麦进行分析，有效地为初步研究中获得的结果提供交叉验证。在植物和谷物发育的整个过程中，通过对绿色组织以及燕麦谷物进行采样进行更多的对照实验，以揭示更深入地了解碳和氮代谢平衡，以及资源分配到脂肪和次生代谢中，也将是令人着迷的。
IntroductionOats (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. -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.ObjectivesOats 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.MethodsFour 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 200kgNha(-1) in a randomised split-plot design. Grain yield, milling quality traits, -glucan, total protein and oil content were assessed. The de-hulled oats (groats) were also subjected to a rapid Ultra High Performance Liquid ChromatographyMass Spectrometry (UHPLC-MS) metabolomic screening approach.ResultsApplication 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. -glucan content significantly increased with nitrogen application. The UHPLC-MS approach has provided a rapid, sub 15min 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 indicating increased TCA cycle turn over, thus providing the plant with a source of energy and reductant power to aid elevated nitrogen assimilation. Elevated nitrogen availability was also directed towards the increased production of nitrogen containing phospholipids. A number of both positive and negative impacts on the metabolism of phenolic compounds that have influence upon the health beneficial value of oats and their products were also observed.ConclusionsAlthough the developed method has broad applicability as a rapid screening method or a rapid metabolite profiling method and in this study has provided valuable metabolic insights, it still must be considered that much greater confidence in metabolite identification, as well as quantitative precision, will be gained by the application of higher resolution chromatography methods, although at a large expense to sample throughput. Follow up studies will apply higher resolution GC (gas chromatography) and LC (reversed phase and HILIC) approaches, oats will be also analysed from across multiple growth locations and growth seasons, effectively providing a cross validation for the results obtained within this preliminary study. It will also be fascinating to perform more controlled experiments with sampling of green tissues, as well as oat grains, throughout the plants and grains development, to reveal greater insight of carbon and nitrogen metabolism balance, as well as resource partitioning into lipid and secondary metabolism.