Metabolites In Maize Research Articles

Catechol acetylglucose: a newly identified benzoxazinoid-regulated defensive metabolite in maize.

An enormous diversity of specialized metabolites is produced in the plant kingdom, with each individual plant synthesizing thousands of these compounds. Previous research showed that benzoxazinoids, the most abundant class of specialized metabolites in maize, also function as signaling molecules by regulating the production callose as a defense response. We searched for additional benzoxazinoid-regulated specialized metabolites, characterized them, examined whether they too function in herbivore protection, and determined how Spodoptera frugiperda (fall armyworm), a prominent maize pest, copes with these metabolites. We identified catechol acetylglucose (CAG) as a benzoxazinoid-regulated metabolite that is produced from salicylic acid via catechol and catechol glucoside. Genome-wide association studies of CAG abundance identified a gene encoding a predicted acetyltransferase. Knockout of this gene resulted in maize plants that lack CAG and over-accumulate catechol glucoside. Upon tissue disruption, maize plants accumulate catechol, which inhibits S. frugiperda growth. Analysis of caterpillar frass showed that S. frugiperda detoxifies catechol by glycosylation, and the efficiency of catechol glycosylation was correlated with S. frugiperda growth on a catechol-containing diet. Thus, the success of S. frugiperda as an agricultural pest may depend partly on its ability to detoxify catechol, which is produced as a defensive metabolite by maize.

Prediction of secondary metabolites in maize under different nitrogen inputs by hyperspectral sensing and machine learning

Flavonoids are compounds resulting from secondary plant metabolism that provide benefits to human health by food. This study aimed to accuracy of predicting flavonoids in maize plants subjected to different nitrogen rates using hyperspectral reflectance and machine learning (ML) algorithms. The experiment was carried out in randomized blocks in a 4 × 5 factorial design (four N inputs: 0; 30; 60 and 120 % of the recommended N input; and five readings of the reflectance spectra in maize leaves from different vegetative stages: V6, V8, V10, V12 and V14, in four replications, totaling 80 treatments. N rates were applied in the V4 and V8 phenological stages, using urea as the N source. For hyperspectral analysis, four leaves from each treatment were collected and analyzed using a spectroradiometer (FieldSpec 4 HRes, Analytical Spectral Devices), capturing the spectrum in the 350 to 2500 nm range. Subsequently, the leaf samples used in the reflectance readings were dried, ground and subjected to isoflavone quantification, analyzed by ultra-performance liquid chromatography in three repetitions, quantifying daidzein 1 (D1), daidzein 2 (D2), genistein 1 (G1), genistein 2 (G2), and total isoflavones. Data obtained was subjected to machine learning analysis, testing two data set input configurations: wavelengths (WL) and calculated spectral bands (B), and D1, D2, G1, G2 and total isoflavones as output variables. The ML algorithms tested were artificial neural networks (ANN), REPTree (DT), M5P decision tree (M5P), ZeroR (R), Random Forest (RF) and support vector machine (SVM), evaluated according to their performance by the correlation coefficient (r) and mean absolute error (MAE). The results show that the SVM algorithm had the highest accuracy in predicting the variables D1, D2, G1, G2 and total isoflavones, outperforming the other algorithms when WL was used as input in dataset.

Benzoxazinoids Biosynthetic Gene Cluster Identification and Expression Analysis in Maize under Biotic and Abiotic Stresses.

Benzoxazinoids (BXs) are unique bioactive metabolites with protective and allelopathic properties in maize in response to diverse stresses. The production of BXs involves the fine regulations of BXs biosynthetic gene cluster (BGC). However, little is known about whether and how the expression pattern of BGC members is impacted by biotic and abiotic stresses. Here, maize BGC was systemically investigated and 26 BGC gene members were identified on seven chromosomes, for which Bin 4.00-4.01/4.03-4.04/7.02 were the most enriched regions. All BX proteins were clearly divided into three classes and seven subclasses, and ten conserved motifs were further identified among these proteins. These proteins were localized in the subcellular compartments of chloroplast, endoplasmic reticulum, or cytoplasmic, where their catalytic activities were specifically executed. Three independent RNA-sequencing (RNA-Seq) analyses revealed that the expression profiles of the majority of BGC gene members were distinctly affected by multiple treatments, including light spectral quality, low-temperature, 24-epibrassinolide induction, and Asian corn borer infestation. Thirteen differentially expressed genes (DEGs) with high and specific expression levels were commonly detected among three RNA-Seq, as core conserved BGC members for regulating BXs biosynthesis under multiple abiotic/biotic stimulates. Moreover, the quantitative real-time PCR (qRT-PCR) verified that six core conserved genes in BGC were significantly differentially expressed in leaves of seedlings upon four treatments, which caused significant increases in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) content under darkness and wound treatments, whereas a clear decrease in DIMBOA content was observed under low-temperature treatment. In conclusion, the changes in BX metabolites in maize were regulated by BGC gene members in multiple stress presences. Therefore, the identification of key genes associated with BX accumulation under biotic/abiotic stresses will provide valuable gene resources for breeding maize varieties with enhanced capability to adapt to environmental stresses.

Partial Chemical Characterization of Drought-Related Metabolites in Maize Under Drought Stress Conditions

This study investigates the metabolic response of maize genotypes UASBM13 (drought-tolerant) and UASBM10 (drought-sensitive) to drought stress using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Drought stress significantly impacts crop productivity, with potential consequences for global food security. Secondary metabolites produced by maize under drought conditions, provide insights into molecular response. The identified metabolites include sinapic acid, sinapoyl malate, coumaroyl shikimate, caffeoyl shikimate, syriogenin, sinapaldehyde, hydroxy ferulate, naringenin chalcone, and resveratrol. These compounds play crucial roles in antioxidant defence, lignification, and stress-related pathways. The results suggest that UASBM13 exhibits a more robust metabolic response to drought, with higher levels of key metabolites associated with stress tolerance. The findings contribute to our understanding of plant adaptation to environmental stress and provide valuable information for developing drought-tolerant crop varieties to ensure global food security in the context of a changing climate and growing population.

Residue determination and dietary risk assessment of mesotrione, nicosulfuron, atrazine and its four metabolites in maize in China

IntroductionTo improve maize yield in China, multiple herbicides have been simultaneously applied to control more weeds. However, this combined application raises concerns about potential residues and their subsequent risks to human health. Therefore, evaluating the residues and dietary risk of new herbicide formulations is critical for the sustainability of maize production.MethodsUsing UHPLC-MS/MS, we developed quick methods for the determination of residues of mesotrione, nicosulfuron, atrazine and its four metabolites with acceptable accuracy and precision. The limits of quantification (LOQs) were 0.01 mg/kg for mesotrione and atrazine-desethyl-desopropyl, and 0.005 mg/kg for nicosulfuron, atrazine, 6-deisopropyl atrazine, 2 hydroxyatrazine, and deethylatrazine. Field trials were conducted at 12 different locations in China. And the risk quotient (RQ) model was used to evaluate the chronic risk of residues of these herbicides.ResultsThe residues of straw samples were in the ranges of <0.01 mg/kg for mesotrione, <0.005–0.010 mg/kg for nicosulfuron, and <0.037–0.254 mg/kg for accumulated atrazine. In green straw, the observed ranges were <0.01 mg/kg for mesotrione, <0.005–0.007 mg/kg for nicosulfuron, and <0.037–0.211 mg/kg for accumulated atrazine. Meanwhile, for both green and fully-grown maize, residues were <0.01 mg/kg for mesotrione, <0.005 mg/kg for nicosulfuron, and <0.037 mg/kg for accumulated atrazine. And all RQ values of green and fully-grown maize were below 100%.ConclusionBased on the RQ model, the dietary risk of exposure to three herbicides through maize was acceptable by consumers. This study helps guide the rational use of mesotrione, nicosulfuron and atrazine to ensure the safe production of maize and our human health.

Residue and dietary risk assessment of glyphosate, glufosinate-ammonium, and their metabolites in maize and soybean

Residue and dietary risk assessment of glyphosate, glufosinate-ammonium, and their metabolites in maize and soybean

A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize.

Two major groups of specialized metabolites in maize (Zea mays), termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider the physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occur in primary roots and show quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of-function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit. Collectively, these results demonstrate dolabralexin biosynthesis via ZmKSL4 as a committed pathway node biochemically separating kauralexin and dolabralexin metabolism, and suggest an interactive role of maize dolabralexins in plant vigor during abiotic stress.

ZmMYC2s play important roles in maize responses to simulated herbivory and jasmonate.

Both herbivory and jasmonic acid (JA) activate the biosynthesis of defensive metabolites in maize, but the mechanism underlying this remains unclear. We generated maize mutants in which ZmMYC2a and ZmMYC2b, two transcription factor genes important in JA signaling, were individually or both knocked out. Genetic and biochemical analyses were used to elucidate the functions of ZmMYC2 proteins in the maize response to simulated herbivory and JA. Compared with the wild-type (WT) maize, the double mutant myc2ab was highly susceptible to insects, and the levels of benzoxazinoids and volatile terpenes, and the levels of their biosynthesis gene transcripts, were much lower in the mutants than in the WT maize after simulated insect feeding or JA treatment. Moreover, ZmMYC2a and ZmMYC2b played a redundant role in maize resistance to insects and JA signaling. Transcriptome and Cleavage Under Targets and Tagmentation-Sequencing (CUT&Tag-Seq) analysis indicated that ZmMYC2s physically targeted 60% of the JA-responsive genes, even though only 33% of these genes were transcriptionally ZmMYC2-dependent. Importantly, CUT&Tag-Seq and dual luciferase assays revealed that ZmMYC2s transactivate the benzoxazinoid and volatile terpene biosynthesis genes IGPS1/3, BX10/11/12/14, and TPS10/2/3/4/5/8 by directly binding to their promoters. Furthermore, several transcription factors physically targeted by ZmMYC2s were identified, and these are likely to function in the regulation of benzoxazinoid biosynthesis. This work reveals the transcriptional regulatory landscapes of both JA signaling and ZmMYC2s in maize and provides comprehensive mechanistic insight into how JA signaling modulates defenses in maize responses to herbivory through ZmMYC2s.

Genetic mapping of maize metabolites using high-throughput mass profiling

Plant metabolites are the basis of human nutrition and have biological relevance in ecology. Farmers selected plants with favorable characteristics since prehistoric times and improved the cultivars, but without knowledge of underlying mechanisms. Understanding the genetic basis of metabolite production can facilitate the successful breeding of plants with augmented nutritional value. To identify genetic factors related to the metabolic composition in maize, we generated mass profiles of 198 recombinant inbred lines (RILs) and their parents (B73 and Mo17) using direct-injection electrospray ionization mass spectrometry (DLI-ESI MS). Mass profiling allowed the correct clustering of samples according to genotype. We quantified 71 mass features from grains and 236 mass features from leaf extracts. For the corresponding ions, we identified tissue-specific metabolic ‘Quantitative Trait Loci’ (mQTLs) distributed across the maize genome. These genetic regions could regulate multiple metabolite biosynthesis pathways. Our findings demonstrate that DLI-ESI MS has sufficient analytical resolution to map mQTLs. These identified genetic loci will be helpful in metabolite-focused maize breeding. Mass profiling is a powerful tool for detecting mQTLs in maize and enables the high-throughput screening of loci responsible for metabolite biosynthesis.

Simultaneous determination of glyphosate, glufosinate ammonium and their metabolites in maize and soybean by ultra-performance liquid chromatography with tandem mass spectrometry.

Glyphosate and glufosinate ammonium are non-selective, broad spectrum herbicides for controlling grasses in agriculture and forestry. Rapid and sensitive analytical methods for effective monitoring and subsequent risk control of glyphosate and glufosinate ammonium and their metabolites in crops are not available. In this paper, an analytical method for the simultaneous determination of glyphosate, glufosinate ammonium, and their metabolites in maize and soybean by ultra-performance liquid chromatography with tandem mass spectrometry was established. The seven compounds were well separated on an anion exchange analytical column within 10 min, with the mean recoveries of the target compounds ranging from 78.2 to 110.9%. The method showed good linearity (R2 ≥ 0.9819) with a limit of quantification for glyphosate, glufosinate ammonium, and their metabolites of 0.01 mg kg-1 in both maize and soybean. The method simplifies and expedites the sample preparation procedures, overcomes the traditional disadvantages including derivatization, weak retention, and low sensitivity, and has been successfully validated in actual maize and soybean samples.

Comprehensive analysis of multiple mycotoxins and Aspergillus flavus metabolites in maize from Kenyan households

This study assessed the levels of mycotoxins in maize from Kenyan households. Further, local open pollinated maize varieties were compared with commercial hybrids to evaluate which variety is less susceptible to mycotoxin contamination. Four hundred and eighty (n = 480) maize samples were collected in the years 2018–2020 from households in Eastern, Western, Coastal and Lake Victoria regions of Kenya. Liquid chromatography coupled to tandem mass spectrometry was used to detect and quantify 22 mycotoxins, along with 31 Aspergillus flavus metabolites in the samples. Eastern Kenya had the highest aflatoxin (AF) contamination with 75% of samples having AF levels above the Kenyan regulatory limits (10 μg/kg), the highest concentration was 558.1 μg/kg. In Western Kenya, only 18% of samples had concentration levels above the Kenyan regulatory limits for AF with highest sample having 73.3 μg/kg. The Lake Victoria region had the most fumonisins (F) contamination, with 53% of the samples having fumonisin B1 (FB1) < 1000 μg/kg. However, only 20% of the samples surpassed the Kenyan regulatory limit for total fumonisins (2000 μg/kg) with the highest concentration being 13,022 μg/kg. In addition, 21.6% of samples from the Lake Victoria region had zearalenone (ZEN) and deoxynivalenol (DON) above regulatory limits for European countries (1000 μg/kg). Western region had the least A. flavus metabolites contamination (18%) while the Eastern region had the highest incidence of A. flavus metabolites (81%). Among the A. flavus metabolites, cyclopiazonic acid (CPA), beta-cyclopiazonic acid (β CPA), flavacol (FLV) and methylcitreo-isocoumarin (MIC) positively correlated with each other but negatively correlated with the other metabolites. Significant positive co-occurrence was also noted among Fusarium mycotoxins: nivalenol (NIV) positively correlated with DON (r = 0.81), fusarenon-X (FX) (r = 0.81) and ZEN (r = 0.70). Negative correlations were observed between Aspergillus and Fusarium mycotoxins: aflatoxin B1 (AFB1) negatively correlated with FB1 (r = −0.11), FX (r = −0.17) and ZEN (r = −0.20). Local open-pollinated maize varieties (L-opv) were less susceptible to mycotoxin contamination compared to the commercial hybrids (C-hy). This study reveals that Kenyan maize is contaminated with multiple mycotoxins most of which are not regulated in Kenya despite being regulated in other parts of the world. A comprehensive legislation should therefore be put in place to protect the Kenyan public against chronic exposure to these mycotoxins. In addition to high yield, there is a need for commercial hybrid maize breeders to incorporate mycotoxin resistance as an important trait in germplasm improvement in seeds production.

Two years study of Aspergillus metabolites prevalence in maize from the Republic of Serbia

Aspergillus species produce numerous metabolites, including mycotoxins, which prevalence in maize is a significant health and economic issue worldwide. The aim of this study was to investigate the prevalence of Aspergillus metabolites in 458 maize samples collected during 2016 and 2017 in the Republic of Serbia. Samples were analyzed by the liquid chromatography-tandem mass spectrometry method. Among the 32 investigated Aspergillus metabolites 9 and 20 were detected in maize samples collected from 2016 and 2017, respectively. The metabolites with the greatest prevalence in 2016 were kojic acid (12%) and 3-nitropropionic acid (10%), while the most commonly detected metabolites in maize from 2017 were kojic acid (54%), 3-nitropropionic acid (44%), aflatoxin B1 (31%), and averufin (29%). Maize growing season in 2017 was characterized by higher deviations of air temperature and a smaller amount of precipitation, which resulted in a greater prevalence of Aspergillus metabolites in maize from 2017 (65%) in comparison to 2016 (23%). Novelty impact statement The main novelty of this study is achieved through the creation of unique database, based on the analysis of 32 Aspergillus metabolites in the great number of maize samples, collected in two different years in the Republic of Serbia. The created extensive database of Aspergillus metabolites is one of the few from this part of Europe, and it will contribute to significant improvement and upgrading of currently existing knowledge and information in this field.

Fusarium metabolites in maize from regions of Northern Serbia in 2016-2017

ABSTRACT The main objective of this study was to determine the presence of Fusarium metabolites in maize samples collected from different regions of Northern Serbia (Bačka, Banat and Srem) during a period of two years (2016–2017). A total of 458 maize samples were analysed by liquid chromatography-tandem mass spectrometry. A total of 40 metabolites were detected, where 94% of the samples contained at least 5 metabolites. Fumonisins (including B1, B2, B3 and B4), moniliformin and bikaverin were the most frequent (80–98%) Fusarium metabolites in both years. Furthermore, in samples from 2016, fumonisin A1 and A2, deoxynivalenol, deoxynivalenol-3-glucoside, zearalenone, culmorin, 15-hydroxyculmorin, fusapyron, fusaproliferin and aurofusarin were detected with frequencies of 58–80%. Levels of certain Fusarium metabolites in 2016 were higher on average due to increased humidity when compared to 2017, which was characterised by warm and dry conditions.

Unveiling the spatial distribution of aflatoxin B1 and plant defense metabolites in maize using AP-SMALDI mass spectrometry imaging.

In order to cope with the presence of unfavorable compounds, plants can biotransform xenobiotics, translocate both parent compounds and metabolites, and perform compartmentation and segregation at the cellular or tissue level. Such a scenario also applies to mycotoxins, fungal secondary metabolites with a pre-eminent role in plant infection. In this work, we aimed to describe the effect of the interplay between Zea mays (maize) and aflatoxin B1 (AFB1) at the tissue and organ level. To address this challenge, we used atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) to investigate the biotransformation, localization and subsequent effects of AFB1 on primary and secondary metabolism of healthy maize plants, both insitu and from a metabolomics standpoint. High spatial resolution (5µm) provided fine localization of AFB1, which was located within the root intercellular spaces, and co-localized with its phase-I metabolite aflatoxin M2. We provided a parallel visualization of maize metabolic changes, induced in different organs and tissues by an accumulation of AFB1. According to our untargeted metabolomics investigation, anthocyanin biosynthesis and chlorophyll metabolism in roots are most affected. The biosynthesis of these metabolites appears to be inhibited by AFB1 accumulation. On the other hand, metabolites found in above-ground organs suggest that the presence of AFB1 may also activate the biochemical response in the absence of an actual fungal infection; indeed, several plant secondary metabolites known for their antimicrobial or antioxidant activities were localized in the outer tissues, such as phenylpropanoids, benzoxazinoids, phytohormones and lipids.

On-tissue boronic acid derivatization for the analysis of vicinal diol metabolites in maize with MALDI-MS imaging.

Derivatization reactions are commonly used in mass spectrometry to improve analyte signals, specifically by enhancing the ionization efficiency of those compounds. Vicinal diols are one group of biologically important compounds that have been commonly derivatized using boronic acid. In this study, a boronic acid with a tertiary amine was adapted for the derivatization of vicinal diol metabolites in B73 maize tissue cross-sections for mass spectrometry imaging analysis. Using this method, dozens of vicinal diol metabolites were derivatized, effectively improving the signal of those metabolites. Many of these metabolites were tentatively assigned using high-resolution accurate mass measurements. In addition, reaction interference and cross-reactivity with various other functional groups were systematically studied to verify data interpretation.

Temporal Regulation of the Metabolome and Proteome in Photosynthetic and Photorespiratory Pathways Contributes to Maize Heterosis.

Heterosis or hybrid vigor is widespread in plants and animals. Although the molecular basis for heterosis has been extensively studied, metabolic and proteomic contributions to heterosis remain elusive. Here we report an integrative analysis of time-series metabolome and proteome data in maize (Zea mays) hybrids and their inbred parents. Many maize metabolites and proteins are diurnally regulated, and many of these show nonadditive abundance in the hybrids, including key enzymes and metabolites involved in carbon assimilation. Compared with robust trait heterosis, metabolic heterosis is relatively mild. Interestingly, most amino acids display negative mid-parent heterosis (MPH), i.e., having lower values than the average of the parents, while sugars, alcohols, and nucleoside metabolites show positive MPH. From the network perspective, metabolites in the photosynthetic pathway show positive MPH, whereas metabolites in the photorespiratory pathway show negative MPH, which corresponds to nonadditive protein abundance and enzyme activities of key enzymes in the respective pathways in the hybrids. Moreover, diurnally expressed proteins that are upregulated in the hybrids are enriched in photosynthesis-related gene-ontology terms. Hybrids may more effectively remove toxic metabolites generated during photorespiration, and thus maintain higher photosynthetic efficiency. These metabolic and proteomic resources provide unique insight into heterosis and its utilization for high yielding maize and other crop plants.

Variation of Fusarium Free, Masked, and Emerging Mycotoxin Metabolites in Maize from Agriculture Regions of South Africa.

The presence of mycotoxins in cereal grain is a very important food safety issue with the occurrence of masked mycotoxins extensively investigated in recent years. This study investigated the variation of different Fusarium metabolites (including the related regulated, masked, and emerging mycotoxin) in maize from various agriculture regions of South Africa. The relationship between the maize producing regions, the maize type, as well as the mycotoxins was established. A total of 123 maize samples was analyzed by a LC-MS/MS multi-mycotoxin method. The results revealed that all maize types exhibited a mixture of free, masked, and emerging mycotoxins contamination across the regions with an average of 5 and up to 24 out of 42 investigated Fusarium mycotoxins, including 1 to 3 masked forms at the same time. Data obtained show that fumonisin B1, B2, B3, B4, and A1 were the most prevalent mycotoxins and had maximum contamination levels of 8908, 3383, 990, 1014, and 51.5 µg/kg, respectively. Deoxynivalenol occurred in 50% of the samples with a mean concentration of 152 µg/kg (max 1380 µg/kg). Thirty-three percent of the samples were contaminated with zearalenone at a mean concentration of 13.6 µg/kg (max 146 µg/kg). Of the masked mycotoxins, DON-3-glucoside occurred at a high incidence level of 53%. Among emerging toxins, moniliformin, fusarinolic acid, and beauvericin showed high occurrences at 98%, 98%, and 83%, and had maximum contamination levels of 1130, 3422, and 142 µg/kg, respectively. Significant differences in the contamination pattern were observed between the agricultural regions and maize types.

An efficient system composed of maize protoplast transfection and HPLC\u2013MS for studying the biosynthesis and regulation of maize benzoxazinoids

BackgroundInsect herbivory poses a major threat to maize. Benzoxazinoids are important anti-insect secondary metabolites in maize, whose biosynthetic pathway has been extensively studied. However, yet little is known about how benzoxazinoids are regulated in maize, partly due to lack of mutant resources and recalcitrance to genetic transformation. Transient systems based on mesophyll- or cultured cell-derived protoplasts have been exploited in several plant species and have become a powerful tool for rapid or high-throughput assays of gene functions. Nevertheless, these systems have not been exploited to study the regulation of secondary metabolites.ResultsA protocol for isolation of protoplasts from etiolated maize seedlings and efficient transfection was optimized. Furthermore, a 10-min-run-time and highly sensitive HPLC–MS method was established to rapidly detect and quantify maize benzoxazinoids. Coupling maize protoplast transfection and HPLC–MS, we screened a few genes potentially regulating benzoxazinoid biosynthesis using overexpression or silencing by artificial microRNA technology.ConclusionsCombining the power of maize protoplast transfection and HPLC–MS analysis, this method allows rapid screening for the regulatory and biosynthetic genes of maize benzoxazinoids in protoplasts, before the candidates are selected for in planta functional analyses. This method can also be applied to study the biosynthesis and regulation of other secondary metabolites in maize and secondary metabolites in other plant species, including those not amenable to transformation.

Metabolome-Scale Genome-Wide Association Studies Reveal Chemical Diversity and Genetic Control of Maize Specialized Metabolites.

Cultivated maize (Zea mays) has retained much of the genetic diversity of its wild ancestors. Here, we performed nontargeted liquid chromatography-mass spectrometry metabolomics to analyze the metabolomes of the 282 maize inbred lines in the Goodman Diversity Panel. This analysis identified a bimodal distribution of foliar metabolites. Although 15% of the detected mass features were present in >90% of the inbred lines, the majority were found in <50% of the samples. Whereas leaf bases and tips were differentiated by flavonoid abundance, maize varieties (stiff-stalk, nonstiff-stalk, tropical, sweet maize, and popcorn) showed differential accumulation of benzoxazinoid metabolites. Genome-wide association studies (GWAS), performed for 3,991 mass features from the leaf tips and leaf bases, showed that 90% have multiple significantly associated loci scattered across the genome. Several quantitative trait locus hotspots in the maize genome regulate the abundance of multiple, often structurally related mass features. The utility of maize metabolite GWAS was demonstrated by confirming known benzoxazinoid biosynthesis genes, as well as by mapping isomeric variation in the accumulation of phenylpropanoid hydroxycitric acid esters to a single linkage block in a citrate synthase-like gene. Similar to gene expression databases, this metabolomic GWAS data set constitutes an important public resource for linking maize metabolites with biosynthetic and regulatory genes.

Method for Residues Analysis of Isoxaflutole and Its Metabolites in Maize

A rapid and sensitive analytical method for the determination of isoxaflutole and diketonitrile (DKN) residues in maize was established based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The samples were extracted with acetonitrile and purified with octadecylsilane (C₁₈). The mass spectrometer was performed in multi-reaction monitoring mode, and electrospray ionization in negative mode was selected. The isoxaflutole and DKN residues were quantified by the external standard method using the matched standard solution to compensate for matrix effect. The results indicated that the calibration curves of isoxaflutole and DKN were linear in the range of 0.005-0.5mg/L with correlation coefficients of more than 0.9922. When the addition level ranged from 0.01 to 0.1mg/kg the average recoveries of isoxaflutole and DKN in maize, green maize and straw were 95%-108%, and the relative standard deviation (RSD) ranged from 2% to 11%. The limit of quantitation (LOQ) were 0.01 mg/kg in different matrices. This method has many characteristics, such as simple, rapid and accurate, and can be adapted for the confirmation of isoxaflutole and DKN residues in maize samples.