Unravelling the regulating role of phytohormones towards carotenoid metabolism in tomato fruit by using the metabolomic screening possibilities of Orbitrap-MS

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 in the occurrence of tomato fruit carotenoids. 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 (with a representative for each hormonal class), 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 a metabolomic approach. The protocol for extraction of the carotenoids (lutein, zeaxanthin, α-carotene, β-carotene, lycopene) 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 ( 85%) were satisfactory. Additionally, the obtained 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 either a control or drought treatment. The purpose was to trigger a stress response in the tomato plant, resulting in altered phytohormone and increased carotenoid concentration levels. Statistical interpretation of the analytical concentrations, obtained from these fruits, allows to investigate the relation between the concerned components. To this end, suited software programs, including ToxIDTM (profiling), SieveTM (fingerprinting) and SimcaTM (data analysis) have been applied