SE183:/S1/M1

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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
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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 M1
Title GC-TOF-MS
Method Details ID MS1
Sample Amount equivalence of 6 μg fresh weight (FW) of the derivatised sample
Comment


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.

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