Untargeted metabolomic analyses (LC-MS/MS) of Arabidopsis seedlings grown in light/dark cycles or in constant light

Résumé 0

In this study, we performed untargeted metabolic analyses (LC-MS/MS) to produce time-series data in Arabidopsis seedlings. These data will be part of Cyclath, a new database dedicated to the study of diurnal and circadian rhythms, and showed how our data can be used to investigate the impact of light/dark cycle and constant light regimes on metabolite rhythmic patterns. RNA-Seq analyses were also included in the database. Plant growth: Arabidopsis plantlets were grown on the modified MS solid medium in a 12 h photoperiod (light/dark) at 100 µmol m-2 s-1. After 17 days, they were either kept in light/dark cycles (LD) or transferred in constant light (LL). Whole leaves were harvested at 24, 28, 32, 36, 40, 44 and 48 hours after transfer and flash frozen into liquid N2. After grinding, they were separated into 2 aliquots per time point and condition. Polar and semi-polar metabolite extraction: Metabolites were then extracted from 50 mg of leaves fresh weight. 1.5 mL of methanol/ water/ acetone/ TFA, V/V 40/ 32/ 28/ 0.05 % and 200 ng of Apigenin (used as internal standard) were added to each sample. The mixtures were then placed in an ultrasonic bath for 5 min at 25 kHz at 4 °C and then shaken for 30 min at 4ºC using a ThermoMixer™ C (Eppendorf). These samples were then centrifuged at 20 000 g for 20 min. The process of extraction was performed twice with the same sample and the two resulting supernatants were pooled and dried in a rotary evaporator and freeze dryer. The dry pellet was dissolved in 150 µL of water ULC/MS grade (Biosolve) with 10% of ACN ULC/MS grade (biosolve) and filtered throught the whatman filter. Chromatography: A Nucleoshell RP 18 plus reversed-phase column (2 x 100 mm, 2.7 μm; Macherey-Nagel) was used for chromatographic separation. The mobile phases used for the chromatographic separation were (A) 0.1% formic acid in H2O and (B) 0.1% formic acid in acetonitrile. The flow rate was of 400 μL/min and the following gradient was used: 95% of A for 1-min, followed by a linear gradient from 95% A to 80% A from 1 to 3-min, then a linear gradient from 80% A to 75% A from 3 to 8-min, a linear gradient from 75% A to 40% A from 8 to 20-min. 0% of A was hold until 24-min, followed by a linear gradient from 0% A to 95% A from 24 to 27-min. Finally, the column was washed by 30% A at for 3.5min then re-equilibrated for 3.5-min (35-min total run time). Mass spectrometry: Data-dependent acquisition (DDA) methods were used for mass spectrometer data in positive and negative ESI modes using the following parameters: capillary voltage, 4.5kV; nebuliser gas flow, 2.1 bar; dry gas flow, 6 L/min; drying gas in the heated electrospray source temperature, 140ºC. Samples were analysed at 8Hz with a mass range of 100 to 1500 m/z. Stepping acquisition parameters were created to improve the fragmentation profile with a collision RF from 200 to 700 Vpp, a transfer time from 20 to 70 µs and collision energy from 20 to 40 eV. Each cycle included a MS fullscan and 5 MS/MS CID on the 5 main ions of the previous MS spectrum. Data transformation: The .d data files (Bruker Daltonics, Bremen, Germany) were converted to .mzXML format using the MSConvert software (ProteoWizard package 3.0).