UPLC-MS-based metabolite analysis in tomato.

Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 860:129-44. DOI: 10.1007/978-1-61779-594-7_9
Source: PubMed

ABSTRACT Recent advances in the performance of hyphenated technologies based on ultrapressure chromatography and high-sensitivity mass spectrometry have set the stage for a myriad of metabolomics studies in plants and other organisms. In this chapter, we describe the use of a UPLC (Ultraperformance Liquid Chromatography)-qTOF (quadrupole time-of-flight) system for profiling semipolar metabolites in the model fruit plant tomato. An optimized extraction method, instrument parameters and data treatment procedures are provided. The value of UPLC instruments, which use small particle size chromatographic columns, in terms of resolution, separation, and short injection times are presented. When coupled to a TOF mass spectrometer with high resolution and mass accuracy, good dynamic range, and a fast spectral acquisition capacity, this system is most suitable for the extensive profiling of hundreds of plant metabolites.

1 Bookmark
  • [Show abstract] [Hide abstract]
    ABSTRACT: Significant inter- and intraspecific genetic variation exists in duckweed, thus the potential for genome plasticity and manipulation is high. Polyploidy is recognised as a major mechanism of adaptation and speciation in plants. We produced several genome-duplicated lines of Landoltia punctata (Spirodela oligorrhiza) from both whole plants and regenerating explants using a colchicine-based cocktail. These lines stably maintained an enlarged frond and root morphology. DNA ploidy levels determined by florescence-activated cell sorting indicated genome duplication. Line A4 was analysed after 75 biomass doublings. Frond area, fresh and dry weights, rhizoid number and length were significantly increased versus wild type, while the growth rate was unchanged. This resulted in accumulation of biomass 17–20% faster in the A4 plants. We sought to determine if specific differences in gene products are found in the genome duplicated lines. Non-targeted ultra performance LC-quadrupole time of flight mass spectrometry was employed to compare some of the lines and the wild type to seek identification of up-regulated metabolites. We putatively identified differential metabolites in Line A65 as caffeoyl hexoses. The combination of directed genome duplication and metabolic profiling might offer a path for producing stable gene expression, leading to altered production of secondary metabolites.
    Plant Biology 07/2014; · 2.32 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tomato (Solanum lycopersicum) fruit contains significant amounts of bioactive compounds, particularly multiple classes of specialized metabolites. Enhancing the synthesis and accumulation of these substances, specifically in fruits, are central for improving tomato fruit quality (e.g. flavour and aroma) and could aid in elucidate pathways of specialized metabolism. To promote the production of specialized metabolites in tomato fruit, this work expressed under a fruit ripening-specific promoter, E8, a bacterial AroG gene encoding a 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is feedback-insensitive to phenylalanine inhibition. DAHPS, the first enzyme of the shikimate pathway, links between the primary and specialized metabolism derived from aromatic amino acids. AroG expression influenced the levels of number of primary metabolites, such as shikimic acid and aromatic amino acids, as well as multiple volatile and non-volatile phenylpropanoids specialized metabolites and carotenoids. An organoleptic test, performed by trained panellists, suggested that the ripe AroG-expressing tomato fruits had a preferred floral aroma compare with fruits of the wild-type line. These results imply that fruit-specific manipulation of the conversion of primary to specialized metabolism is an attractive approach for improving fruit aroma and flavour qualities as well as discovering novel fruit-specialized metabolites.
    Journal of Experimental Botany 01/2013; · 5.79 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Metabolite composition offers a powerful tool for understanding gene function and regulatory processes. However, metabolomics studies on multicellular organisms have thus far been performed primarily on whole organisms, organs, or cell lines, losing information about individual cell types within a tissue. With the goal of profiling metabolite content in different cell populations within an organ, we used FACS to dissect GFP-marked cells from Arabidopsis roots for metabolomics analysis. Here, we present the metabolic profiles obtained from five GFP-tagged lines representing core cell types in the root. Fifty metabolites were putatively identified, with the most prominent groups being glucosinolates, phenylpropanoids, and dipeptides, the latter of which is not yet explored in roots. The mRNA expression of enzymes or regulators in the corresponding biosynthetic pathways was compared with the relative metabolite abundance. Positive correlations suggest that the rate-limiting steps in biosynthesis of glucosinolates in the root are oxidative modifications of side chains. The current study presents a work flow for metabolomics analyses of cell-type populations.
    Proceedings of the National Academy of Sciences 03/2013; · 9.81 Impact Factor


Available from