SE183:/MS1

Sample Set Information

ID	TSE1341
Title	Metabolomics reveals comprehensive reprogramming involving two independent metabolic responses of Arabidopsis to UV-B light.
Description	Because of ever-increasing environmental deterioration it is likely that the influx of UV-B radiation (280-320 nm) will increase as a result of the depletion of stratospheric ozone. Given this fact it is essential that we better understand both the rapid and the adaptive responses of plants to UV-B stress. Here, we compare the metabolic responses of wild-type Arabidopsis with that of mutants impaired in flavonoid (transparent testa 4, tt4; transparent testa 5, tt5) or sinapoyl-malate (sinapoylglucose accumulator 1, sng1) biosynthesis, exposed to a short 24-h or a longer 96-h exposure to this photo-oxidative stress. In control experiments we subjected the genotypes to long-day conditions as well as to 24- and 96-h treatments of continuous light. Following these treatments we evaluated the dynamic response of metabolites including flavonoids, sinapoyl-malate precursors and ascorbate, which are well known to play a role in cellular protection from UV-B stress, as well as a broader range of primary metabolites, in an attempt to more fully comprehend the metabolic shift following the cellular perception of this stress. Our data reveals that short-term responses occur only at the level of primary metabolites, suggesting that these effectively prime the cell to facilitate the later production of UV-B-absorbing secondary metabolites. The combined results of these studies together with transcript profiles using samples irradiated by 24-h UV-B light are discussed in the context of current models concerning the metabolic response of plants to the stress imposed by excessive UV-B irradiation.
Authors	Kusano, M., Tohge, T., Fukushima, A., Kobayashi, M., Hayashi, N., Otsuki, H., Kondou, Y., Goto, H.,Kawashima, M., Matsuda, F., Niida, R., Matsui, M., Saito, K. and Fernie, A. R.
Reference	Plant J. 2011 Jul;67(2):354-69. doi: 10.1111/j.1365-313X.2011.04599.x
Comment

PubMed

Analytical Method Details Information

ID	MS1
Title	GC-TOF-MS
Instrument	GC:Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) MS:Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA)
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	BioSource amount: The harvested samples were weighed and then each biological sample was put in a 2-ml tube with 5 mm Zirconia beads to be used for metabolite profiling. For GC-TOF-MS analysis: an equivalence of 6 μg fresh weight (FW) of the derivatised sample was injected. Sample processing and extraction: The frozen sample in a 2 ml tube was extracted with a concentration of 25 mg FW of tissues per ml extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds using a Retsch mixer mill MM310 (Retsch, Haan, Germany) at a frequency of 30 Hz for 3 min at 4°C. After centrifugation, a 200-μl of the supernatant was used for GC-TOF-MS analysis, while a 100-μl of the supernatant for LC-q-TOF-MS analysis. Extraction and derivatization for GC-TOF-MS analysis: Each sample was extracted with a concentration of 25 mg FW of tissues per µl extraction medium (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds ([2H4]-succinic acid, [13C5,15N]-glutamic acid, [2H7]-cholesterol, [13C3]-myristic acid, [13C5]-proline, [13C12]-sucrose, [13C4]-hexadecanoic acid, [2H4]-1,4-butanediamine, [2H6]-2-hydoxybenzoic acid, and [13C6]-glucose) using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope compound was adjusted to a final concentration of 15 ng/µl for each 1-µl injection. After centrifugation for 5 min at 15,100 × g, a 200-µl aliquot of the supernatant was drawn and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA). For methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. Thirty µl of n-heptane was added following silylation. All the derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen. GC-TOF-MS conditions One microliter of each sample was injected in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwin-gen, Switzerland) into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) equipped with a 30 m × 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-μl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis. Helium was used as the carrier gas at a constant flow rate of 1 ml/min. The temperature program for metabolome analysis started with a 2-min isothermal step at 80 °C and this was followed by temperature ramping at 30 °C to a final temperature of 320 °C, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 °C, respectively. Ions were generated by a 70-eV electron beam at an ionization current of 2.0 mA. The acceleration voltage was turned on after a solvent delay of 235 s. Data acquisition was performed on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra/s in the mass range of a mass-to-charge ratio of m/z = 60–800. Alkane standard mixtures (C8–C20 and C21–C40) were purchased from Sigma–Aldrich (Tokyo, Japan) and were used for calculating the retention index (RI) (Schauer et al., 2005, Wagner et al., 2003). For quality control, we injected methylstearate in every 6 samples.
Comment_of_details