High Resolution Mass Spectrometry-based Plant Metabolites Analysis

Plant metabolites have diverse chemical and physical properties,they are end products of genes regulation that reflect the physiological condition within the plant.Comprehensive profiling of plant metabolites of various mutants or under different physiological/stress conditions can provide a perspective view of regulatory network of plants.It is of great importance to improve resistance of plants to adverse environment,as well as the yield and quality of crops.In this study,high resolution mass spectrometry-based plant metabolomics were used to elucidate the roles of plant metabolites.Methods for simultaneous quantitation of 6 phytohormones and 40 amino acids were established.Combined with gene expression analysis,the effects of JAs on Arabidopsis metabolome were investigated.In addition,the metabolome of teosinte and maize were compared and the genetic variation of metabolites during maize domestication was analyzed.Researches on the functions and interaction networks of phytohormones rely on accurate determination of phytohormones,preferably in a simultaneous manner.Endogenous phytohormones have diverse chemical structures and exist in extremely low concentrations in plant tissue.In this work,a simple and efficient method for simultaneous quantitation of multiple hormones in plant samples was developed using liquid-liquid extraction and isotope dilution.Ultra high performance liquid chromatography and high resolution mass spectrometry(UPLC-HRMS)with parallel reaction monitoring were used to ensure specificity and sensitivity of the method.Accuracy,sensitivity,linearity,recovery and precision of the method were validated and can meet the requirement of the analysis of real samples.The spatial and temporal changes of phytohormones in Arabidopsis were determined with the developed method.Free amino acids and related compounds in plants have significant impacts on food,medicine,and agriculture industries.Fast and simultaneous quantitation of free amino acids and related compounds based on reversed phase chromatography was limited by low retention of these polar compounds,limited peak capacity of the chromatographic column and low detection sensitivity.Here,a high throughput method for simultaneous profiling of 40 amino acids and related compounds in plant samples by the use of pre-column derivatization and ultra performance liquid chromatography coupled with high resolution mass spectrometry was established.Specificity,linearity,sensitivity,recovery and reproducibility of the method were validated.With the advantage of fast,efficient and highly sensitive,this method was applied to profile analysis of amino acids and related compounds in vegetables for nutritional assessment.The results showed that this method was robust to adapt the complex matrix of plant materials and can be expanded to the analysis of juices,fruits and other related samples.Jasmonates(JAs)play important roles in plant growth,development and defense.Comprehensive metabolomics profiling of plants under JA treatment provides insights into the interaction and regulation network of plant hormones.Here,high resolution mass spectrometry based metabolomics approach was applied to Arabidopsis wild type and JA synthesis deficiency mutant opr3.The effects of exogenous MeJA treatment on the metabolites of opr3 were investigated.More than 10000 ion signals were detected and more than 2000 signals showed significant variation in different genotypes and treatment groups.Multivariate statistic analyses were performed and a differential compound library containing 174 metabolites with high resolution precursor ion-product ions pairs was obtained.Classification and pathway analysis of 109 identified compounds in this library showed that glucosinolates and tryptophan metabolism,amino acids and small peptides metabolism,lipid metabolism,especially fatty acyls metabolism,were impacted by endogenous JA deficiency and exogenous MeJA treatment.These results were further verified by quantitative reverse transcription PCR(RT-qPCR)analysis of 21 related genes involved in the metabolism of glucosinolates,tryptophan and a-linolenic acid pathways.The results would greatly enhance our understanding of the biological functions of JA.Maize is one of the most important corps in the world.It is of great significance to study the variation of metabolites during the domestication of maize.In this work,the metabolome of 30 teosinte and 40 maize lines were analyzed using UPLC-HRMS.Statistic analyses showed metabolome variation between teosinte and maize.Thousands of differential metabolites were found and 97 were identified.Metabolome of 624 recombinant inbred population derived from teosinte and W22 were also analyzed and 299 metabolites with QTL mapping results were identified.These metabolites belong to different categories were involved in several important metabolic pathways.Future genetic analysis of the metabolites and cloning of related genes will provide guidance for maize genetic improvement.