Intact Glucosinolate Analysis in Plant Extracts by Programmed Cone Voltage Electrospray LC/MS: Performance and Comparison with LC/MS/MS Methods

Mellon, Fred A.; Bennett, Richard N.; Holst, Birgit; Williamson, Gary

doi:10.1006/abio.2002.5677

Cited by 115 publications

(113 citation statements)

References 25 publications

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“…The reaction was started by the addition of PAPS, incubated for 30 min at 30°C, and stopped by incubation for 20 min at 95°C. The formation of intact allyl GLS was analyzed by high performance liquid chromatography according to Mellon et al (17). Separation was achieved with a gradient of 0.1% trifluoroacetic acid and acetonitrile with 0.1% trifluoroacetic acid on an RP-C18 column (Supelcosil LC18-DB, 250 ϫ 4.6 mm, 5 m).…”

Section: Methodsmentioning

confidence: 99%

“…Separation was achieved with a gradient of 0.1% trifluoroacetic acid and acetonitrile with 0.1% trifluoroacetic acid on an RP-C18 column (Supelcosil LC18-DB, 250 ϫ 4.6 mm, 5 m). The product was identified by its retention time, absorption spectrum, and mass spectrum as described by Mellon et al (17). Quantification of the product formation was done with a calibration curve prepared using authentic sinigrin.…”

Section: Methodsmentioning

confidence: 99%

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Elucidation of Gene-to-Gene and Metabolite-to-Gene Networks inArabidopsis by Integration of Metabolomics andTranscriptomics

Hirai

Klein

Fujikawa

et al. 2005

Journal of Biological Chemistry

444

368

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Since the completion of genome sequences of model organisms, functional identification of unknown genes has become a principal challenge in biology. Postgenomics sciences such as transcriptomics, proteomics, and metabolomics are expected to discover gene functions. This report outlines the elucidation of gene-togene and metabolite-to-gene networks via integration of metabolomics with transcriptomics and presents a strategy for the identification of novel gene functions. Metabolomics and transcriptomics data of Arabidopsis grown under sulfur deficiency were combined and analyzed by batch-learning self-organizing mapping. A group of metabolites/genes regulated by the same mechanism clustered together. The metabolism of glucosinolates was shown to be coordinately regulated. Three uncharacterized putative sulfotransferase genes clustering together with known glucosinolate biosynthesis genes were candidates for involvement in biosynthesis. In vitro enzymatic assays of the recombinant gene products confirmed their functions as desulfoglucosinolate sulfotransferases. Several genes involved in sulfur assimilation clustered with O-acetylserine, which is considered a positive regulator of these genes. The genes involved in anthocyanin biosynthesis clustered with the gene encoding a transcriptional factor that up-regulates specifically anthocyanin biosynthesis genes. These results suggested that regulatory metabolites and transcriptional factor genes can be identified by this approach, based on the assumption that they cluster with the downstream genes they regulate. This strategy is applicable not only to plant but also to other organisms for functional elucidation of unknown genes.In the era of post-genomics, a systematic and comprehensive understanding of the complex events of life is a great concern in biology. The first step in this process is to identify all gene functions and gene-to-gene networks as the components of the system, the whole events of life. The metabolome is the final product of a series of gene actions. Hence, metabolomics has a potential to elucidate gene functions and networks, especially when integrated with transcriptomics. A promising approach is pair-wise transcript-metabolite correlation analysis, which can reveal unexpected correlations and bring to light candidate genes for modifying the metabolite content (1). Gene functions involved in the specific pathway of interest have been identified by the integration of transcript and targeted metabolic profiling in experimental systems where the pathway was activated (2-6). However, up to now, no gene function has been identified by non-targeted analyses of the transcriptome and metabolome. In this report, we analyzed the non-targeted metabolome and transcriptome of a model plant Arabidopsis under sulfur (S) 1 deficiency whose genome sequencing has been completed. Our strategy for integrated analyses using batch-learning-selforganizing mapping (BL-SOM) (7-9) enabled the identification of gene-to-gene and metabolite-to-gene networks and new gene fun...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Elucidation of Gene-to-Gene and Metabolite-to-Gene Networks inArabidopsis by Integration of Metabolomics andTranscriptomics

Hirai

Klein

Fujikawa

et al. 2005

Journal of Biological Chemistry

444

368

View full text Add to dashboard Cite

show abstract

“…Glucosinolates were analyzed by liquid chromatography-mass spectrometry modifying the methods described by Mellon et al (2002). Ten microliters of sample was applied to the Acquity UPLC system (Waters) and separated on SunFire C18 column (150 3 2.1-mm diameter; Waters) under a linear gradient elution program with solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in methanol): 0 to 2% solvent B (8 min), 2 to 30% solvent B (12 min), 30 to 100% solvent B (18 min), 100% solvent B (7 min), and 100% solvent A (5 min).…”

Section: Metabolite Analysismentioning

confidence: 99%

ArabidopsisSLIM1 Is a Central Transcriptional Regulator of Plant Sulfur Response and Metabolism

et al. 2006

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Sulfur is an essential macronutrient required for plant growth. To identify key transcription factors regulating the sulfur assimilatory pathway, we screened Arabidopsis thaliana mutants using a fluorescent reporter gene construct consisting of the sulfur limitation-responsive promoter of the SULTR1;2 sulfate transporter and green fluorescent protein as a background indicator for monitoring plant sulfur responses. The isolated mutant, sulfur limitation1 (slim1), was unable to induce SULTR1;2 transcripts under low-sulfur (-S) conditions. Mutations causing the sulfur limitation responseless phenotypes of slim1 were identified in an EIL family transcription factor, ETHYLENE-INSENSITIVE3-LIKE3 (EIL3), whose functional identity with SLIM1 was confirmed by genetic complementation. Sulfate uptake and plant growth on -S were significantly reduced by slim1 mutations but recovered by overexpression of SLIM1. SLIM1 functioned as a central transcriptional regulator, which controlled both the activation of sulfate acquisition and degradation of glucosinolates under -S conditions. Metabolite analysis indicated stable accumulation of glucosinolates in slim1 mutants, even under -S conditions, particularly in the molecular species with methylsulfinylalkyl side chains beneficial to human health. Overexpression of SLIM1 and its rice (Oryza sativa) homologs, but no other EIL genes of Arabidopsis, restored the sulfur limitation responseless phenotypes of slim1 mutants, suggesting uniqueness of the SLIM1/EIL3 subgroup members as sulfur response regulators. INTRODUCTIONModern agriculture requires adequate fertilization of sulfur to achieve maximum crop yield and performances. Plants use sulfate, the oxidized form of sulfur existing in the soil, as a sulfur source (Crawford et al., 2000;Leustek et al., 2000;Saito, 2004). By contrast, animals, including humans, require sulfur-containing amino acids and proteins as dietary sulfur sources because of their inability to assimilate sulfate into Cys and Met. Significance of plant sulfate assimilatory pathway is manifested by its ability to fill this metabolic gap in the global sulfur cycle in nature (Crawford et al., 2000). In addition to its basic nutritional importance, sulfur is present in numbers of plant metabolites representing important biological activities as redox controllers, vitamins, coenzymes, flavors, and defense chemicals (Crawford et al., 2000;Leustek et al., 2000;Saito, 2004;Grubb and Abel, 2006;Halkier and Gershenzon, 2006). Activation of sulfate transport systems is critical for plant growth under a low-sulfur (-S) environment. When the soil environment is inadequately fertilized with sulfate, plants will sustain their growth by increasing the capacities of sulfate uptake systems in roots (Clarkson et al., 1983;Deane-Drummond, 1987;Smith et al., 1995Smith et al., , 1997. In Arabidopsis thaliana, highaffinity sulfate transporters that facilitate the initial uptake of sulfate serve this purpose (Takahashi et al., 2000;Vidmar et al., 2000;Shibagaki et al., 2002;Yoshimoto et a...

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“…Glucosinolates were extracted by 70% v/v methanol following Mellon et al (2002) and Wathelet et al (2004). Analysis of glucosinolates was then carried out using a HPLC/DAD/ESI-MS (HP 1100 series, Agilent Technologies) with a C-18 Nucleodur sphinx reversed-phase column Primers sets used in the first PCR: F203a and R1494 (Alphaproteobacteria), F948b and R1494 (Betaproteobacteria), gF382 and gR946, gR1363 (Gammaproteobacteria), Acid31 and S10 (Acidobacteria), Ar3F and Ar9R (archaea), NS0 and EF3 (fungi).…”

Section: Analysis Of Glucosinolates and Derivativesmentioning

confidence: 99%

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

et al. 2009

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A specificity of Brassicaceous plants is the production of sulphur secondary metabolites called glucosinolates that can be hydrolysed into glucose and biocidal products. Among them, isothiocyanates are toxic to a wide range of microorganisms and particularly soil-borne pathogens. The aim of this study was to investigate the role of glucosinolates and their breakdown products as a factor of selection on rhizosphere microbial community associated with living Brassicaceae. We used a DNA-stable isotope probing approach to focus on the active microbial populations involved in root exudates degradation in rhizosphere. A transgenic Arabidopsis thaliana line producing an exogenous glucosinolate and the associated wild-type plant associated were grown under an enriched 13 CO 2 atmosphere in natural soil. DNA from the rhizospheric soil was separated by density gradient centrifugation. Bacterial (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Acidobacteria), Archaea and fungal community structures were analysed by DGGE fingerprints of amplified 16S and 18S rRNA gene sequences. Specific populations were characterized by sequencing DGGE fragments. Roots of the transgenic plant line presented an altered profile of glucosinolates and other minor additional modifications. These modifications significantly influenced microbial community on roots and active populations in the rhizosphere. Alphaproteobacteria, particularly Rhizobiaceae, and fungal communities were mainly impacted by these Brassicaceous metabolites, in both structure and composition. Our results showed that even a minor modification in plant root could have important repercussions for soil microbial communities.

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Intact Glucosinolate Analysis in Plant Extracts by Programmed Cone Voltage Electrospray LC/MS: Performance and Comparison with LC/MS/MS Methods

Cited by 115 publications

References 25 publications

Elucidation of Gene-to-Gene and Metabolite-to-Gene Networks inArabidopsis by Integration of Metabolomics andTranscriptomics

Elucidation of Gene-to-Gene and Metabolite-to-Gene Networks inArabidopsis by Integration of Metabolomics andTranscriptomics

ArabidopsisSLIM1 Is a Central Transcriptional Regulator of Plant Sulfur Response and Metabolism

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

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