SE183:/MS2

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	MS2
Title	LC-q-TOF-MS
Instrument	HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier
Instrument Type	UPLC-QTOF-MS
Ionization	ESI
Ion Mode	positive and negative
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 LC-q-TOF-MS analysis: an equivalence of 300 μg FW of the samples 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 for LC-q-TOF-MS analysis: a 100-μl of the supernatant of each sample in a 1.5 ml tube was used for LC-q-TOF-MS analysis. One hundred-μl of chloroform and one hundred-μl of water containing two reference compounds (0.5 mg/l flavonol-2’-sulfonic acid and 1.0 mg/l ampicilin) at 4°C was added to the tube and then shaken. After 10 min at 4°C, extracts were centrifuged for 3 min at 1,200 x g. The supernatant was transferred and then evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA). The extracts were dissolved by 25 μl of 20% aqueous methanol containing 0.5 mg l−1 lidocaine and d-camphor sulfonic acid (final concentration: 1 mg FW per 10-μl sample). LC-q-TOF-MS conditions: After filtration of the extracts (Ultrafree-MC, 0.2 μm pore size; Millipore), the sample extracts (3 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier). The analytical conditions were as follows. HPLC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μm, length 2.0 × 100 mm, Waters); solvent system, acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program, 1 : 99 v/v at 0 min, 1 : 99 v/v at 0.1 min, 99.5 : 0.5 at 15.5 min, 99.5 : 0.5 at 17.0 min, 1 : 99 v/v at 17.1 min and 1 : 99 at 20 min; flow rate, 0.3 ml min−1; temperature, 38°C; MS detection: capillary voltage, +3.0 keV; cone voltage, 23 V for positive mode and 35 V for negative mode; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l h−1; desolvation gas flow, 800 l/ h; collision energy, 2 V for positive mode and 5 V for negative mode ; detection mode, scan (m/z 100–2000; dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 19.5 min in a single run. The data were recorded using MassLynx version 4.1 software (Waters).
Comment_of_details