SE183:/S1/M2

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

Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	BioSource Species: Arabidopsis thaliana L. cv. Columbia ecotype 0 (wild-type) Genotype: Wild-type, transparent-testa-4 [tt4] (Shikazono et al., 2001), transparent-testa-5 [tt5] (SALK_034145) and sinapoylglucose-accumulator-1 [sng1] (Lorenzen et al., 1996) in Col-0 background Organ: Aerial regions Organ specification: 18-day-old aerial parts were harvested. Growth condition: The sterilized seeds were stratified at 5°C for 2 d, and were successively sown on Murashige and Skoog (MS) medium (Wako, Osaka, Japan) containing 1% sucrose. These four genotypes were grown, side-by-side, under highly controlled growth conditions, under five independent environmental regimes. The chosen conditions were; (i) growth for 18 days under typical long day conditions (16h light/ 8h dark photoperiod; LC), (ii) growth for 17 days under typical long day conditions with 8 h additional light on day 18 (continuous light 1; CO1), (iii) growth for 17 days under typical long day conditions with 24-h ultraviolet light on day 18 (ultraviolet light 1; UV1), (iv) growth for 14 days under typical long day conditions followed by four days (96h) of continuous light (continuous light 4; CO4) and (v) growth for 14 days under typical long day conditions followed by four days of ultraviolet light (ultraviolet light 4; UV4). Plants were cultivated in controlled growth chambers (SANYO; MLR-350) at 22°C in 16-h light and 8-h dark conditions for 14 days (for CO4 and UV4), 17 days (for CO1 and UV1) and 18 days (for LC). The aerial regions were harvested, 6 h after the onset of the bright phase under each light treatment. Among these harvested plants, the plants that had fresh weight over 10 mg were used for metabolite profiling (9-11 different biological replicates for each condition). Experimental condition: See the growth conditions. Light condition: 50 μmol m-2 s-1 for LC, 60 μmol m-2 s-1 for CO1 and CO4; 830 mW m-2 s-1 for UV1 and UV 4 irradiated by narrow-band (312 nm) ultraviolet-B light (CSL-30B, COSMO BIO). Growth plot design: Plates were randomized every third day in each growth chamber. Sampling and sampling date: The aerial regions were harvested in February 2007 (5th, 6th and 7th February, 2007) for metabolite profiling, in July and August 2009 for qRT-PCR and microarray analysis. Metabolism quenching method: All the plant materials were frozen immediately (within 30 sec after sampling) in liquid nitrogen to quench the enzymatic activity. Samples were stored at -80°C until use.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M2
Title	LC-q-TOF-MS
Method Details ID	MS2
Sample Amount	equivalence of 300 μg FW of the samples
Comment

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