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Unravelling the phytohormonal status and carotenoid profile of tomato plants by using metabolomics, including generic extraction and UHPLC-Orbitrap-MS

Lieven Van Meulebroek (UGent) , Julie Vanden Bussche (UGent) , Kathy Steppe (UGent) and Lynn Vanhaecke (UGent)
Author
Organization
Project
IWT 101188
Abstract
Carotenoids are important secondary metabolites, which are mainly synthesized by plants, algae and certain types of bacteria and fungi. In epidemiological and clinical studies, associations were found between the intake of the concerned dietary compounds and the prevalence of chronic-degenerative diseases. Because of these beneficial health effects, carotenoids are intensively studied to better understand amongst others their partitioning and metabolism in plants. In this context, phytohormones might fulfil an important role since these compounds have a regulatory function in various plant processes. This study aimed for a better understanding of carotenoids’ metabolism by investigating the regulating role of phytohormones towards the occurrence of carotenoids in tomato fruit. The realization of the outlined objective completely fits into the food omics concept, which is intended to be a global discipline that includes all of the emerging working areas in which food, analytical techniques and bioinformatics are combined. Completing this aim required the development of analytical methods for quantification of both carotenoids and phytohormones, present in tomato plant tissue. For the extraction of phytohormones, the developed protocol included solid liquid extraction with Bieleski solvent (methanol/water/formic acid), a purification step using a 30 kDa Amicon centrifugal filter unit, and an enrichment phase. Extracts were analyzed using UHPLC-Orbitrap ExactiveTM MS. The high resolution (up to 100,000 FWHM) and the full-scan principle of the applied mass spectrometer were particularly suited to realize the metabolomic approach. The protocol for extraction of the carotenoids was developed by means of a factorial D-optimal design and consisted of liquid-liquid extraction, using methyl-tert-butyl ether and methanol. Extracts were also analyzed by Orbitrap ExactiveTM MS. For chromatographic separation, a C30 HPLC-column was used. Both methods were successfully validated since linearity (R2 > 0.99), repeatability (< 15%), within-laboratory reproducibility (< 20%), specificity and recovery (> 85%) were in general compliant with the requirements of CD 2002/657/EC. Additionally, in comparison with literature, determined LOD (S/N ≥ 3) and LOQ (S/N ≥ 10) values were good for all targeted analytes. Subsequently, an experiment was set up in which tomato plants were subjected to different salt stress levels. The purpose was to unravel the influence of salt stress on phytohormonal plant status and related tomato fruit antioxidant content. To this end, efficient interpretation of the full-scan data will be realized by using suited software programs, including ToxIDTM (profiling), SieveTM (fingerprinting) and SimcaTM (data analysis). The different aspects of the described food omics approach will be discussed.
Keywords
Metabolomics, Bio-informatics, Orbitrap-MS, Phytohormones, Carotenoids

Citation

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Chicago
Van Meulebroek, Lieven, Julie Vanden Bussche, Kathy Steppe, and Lynn Vanhaecke. 2014. “Unravelling the Phytohormonal Status and Carotenoid Profile of Tomato Plants by Using Metabolomics, Including Generic Extraction and UHPLC-Orbitrap-MS.” In 13th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) and 3rd International Symposium on Hyphenated Techniques for Sample Preparation (HTSP-3), Book of Abstracts.
APA
Van Meulebroek, L., Vanden Bussche, J., Steppe, K., & Vanhaecke, L. (2014). Unravelling the phytohormonal status and carotenoid profile of tomato plants by using metabolomics, including generic extraction and UHPLC-Orbitrap-MS. 13th International symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) and 3rd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-3), Book of abstracts. Presented at the 13th International symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) ; 3rd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-3).
Vancouver
1.
Van Meulebroek L, Vanden Bussche J, Steppe K, Vanhaecke L. Unravelling the phytohormonal status and carotenoid profile of tomato plants by using metabolomics, including generic extraction and UHPLC-Orbitrap-MS. 13th International symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) and 3rd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-3), Book of abstracts. 2014.
MLA
Van Meulebroek, Lieven, Julie Vanden Bussche, Kathy Steppe, et al. “Unravelling the Phytohormonal Status and Carotenoid Profile of Tomato Plants by Using Metabolomics, Including Generic Extraction and UHPLC-Orbitrap-MS.” 13th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) and 3rd International Symposium on Hyphenated Techniques for Sample Preparation (HTSP-3), Book of Abstracts. 2014. Print.
@inproceedings{4252138,
  abstract     = {Carotenoids are important secondary metabolites, which are mainly synthesized by plants, algae and certain types of bacteria and fungi. In epidemiological and clinical studies, associations were found between the intake of the concerned dietary compounds and the prevalence of chronic-degenerative diseases. Because of these beneficial health effects, carotenoids are intensively studied to better understand amongst others their partitioning and metabolism in plants. In this context, phytohormones might fulfil an important role since these compounds have a regulatory function in various plant processes. 
This study aimed for a better understanding of carotenoids{\textquoteright} metabolism by investigating the regulating role of phytohormones towards the occurrence of carotenoids in tomato fruit. The realization of the outlined objective completely fits into the food omics concept, which is intended to be a global discipline that includes all of the emerging working areas in which food, analytical techniques and bioinformatics are combined. Completing this aim required the development of analytical methods for quantification of both carotenoids and phytohormones, present in tomato plant tissue. For the extraction of phytohormones, the developed protocol included solid liquid extraction with Bieleski solvent (methanol/water/formic acid), a purification step using a 30 kDa Amicon\unmatched{f8e8} centrifugal filter unit, and an enrichment phase. Extracts were analyzed using UHPLC-Orbitrap ExactiveTM MS. The high resolution (up to 100,000 FWHM) and the full-scan principle of the applied mass spectrometer were particularly suited to realize the metabolomic approach. The protocol for extraction of the carotenoids was developed by means of a factorial D-optimal design and consisted of liquid-liquid extraction, using methyl-tert-butyl ether and methanol. Extracts were also analyzed by Orbitrap ExactiveTM MS. For chromatographic separation, a C30 HPLC-column was used. Both methods were successfully validated since linearity (R2 {\textrangle} 0.99), repeatability ({\textlangle} 15\%), within-laboratory reproducibility ({\textlangle} 20\%), specificity and recovery ({\textrangle} 85\%) were in general compliant with the requirements of CD 2002/657/EC.  Additionally, in comparison with literature, determined LOD (S/N \ensuremath{\geq} 3) and LOQ (S/N \ensuremath{\geq} 10) values were good for all targeted analytes. Subsequently, an experiment was set up in which tomato plants were subjected to different salt stress levels. The purpose was to unravel the influence of salt stress on phytohormonal plant status and related tomato fruit antioxidant content. To this end, efficient interpretation of the full-scan data will be realized by using suited software programs, including ToxIDTM (profiling), SieveTM (fingerprinting) and SimcaTM (data analysis). The different aspects of the described food omics approach will be discussed.},
  author       = {Van Meulebroek, Lieven and Vanden Bussche, Julie and Steppe, Kathy and Vanhaecke, Lynn},
  booktitle    = {13th International symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) and 3rd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-3), Book of abstracts},
  language     = {eng},
  location     = {Brugge, Belgium},
  title        = {Unravelling the phytohormonal status and carotenoid profile of tomato plants by using metabolomics, including generic extraction and UHPLC-Orbitrap-MS},
  year         = {2014},
}