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Sample Set Information

ID TSE1302
Title Effects of Combined Low Glutathione with Mild Oxidative and Low Phosphorus Stress on the Metabolism of Arabidopsis thaliana
Description Plants possess highly sensitive mechanisms that monitor environmental stress levels for a dose-dependent fine-tuning of their growth and development. Differences in plant responses to severe and mild abiotic stresses have been recognized. Although many studies have revealed that glutathione can contribute to plant tolerance to various environmental stresses, little is known about the relationship between glutathione and mild abiotic stress, especially the effect of stress-induced altered glutathione levels on the metabolism. Here, we applied a systems biology approach to identify key pathways involved in the gene-to-metabolite networks perturbed by low glutathione content under mild abiotic stress in Arabidopsis thaliana. We used glutathione synthesis mutants (cad2-1 and pad2-1) and plants overexpressing the gene encoding γ-glutamylcysteine synthetase, the first enzyme of the glutathione biosynthetic pathway. The plants were exposed to two mild stress conditions—oxidative stress elicited by methyl viologen and stress induced by the limited availability of phosphate. We observed that the mutants and transgenic plants showed similar shoot growth as that of the wild-type plants under mild abiotic stress. We then selected the synthesis mutants and performed multi-platform metabolomics and microarray experiments to evaluate the possible effects on the overall metabolome and the transcriptome. As a common oxidative stress response, several flavonoids that we assessed showed overaccumulation, whereas the mild phosphate stress resulted in increased levels of specific kaempferol- and quercetin-glycosides. Remarkably, in addition to a significant increased level of sugar, osmolytes, and lipids as mild oxidative stress-responsive metabolites, short-chain aliphatic glucosinolates over-accumulated in the mutants, whereas the level of long-chain aliphatic glucosinolates and specific lipids decreased. Coordinated gene expressions related to glucosinolate and flavonoid biosynthesis also supported the metabolite responses in the pad2-1 mutant. Our results suggest that glutathione synthesis mutants accelerate transcriptional regulatory networks to control the biosynthetic pathways involved in glutathione-independent scavenging metabolites, and that they might reconfigure the metabolic networks in primary and secondary metabolism, including lipids, glucosinolates, and flavonoids. This work provides a basis for the elucidation of the molecular mechanisms involved in the metabolic and transcriptional regulatory networks in response to combined low glutathione content with mild oxidative and nutrient stress in A. thaliana.
Authors Fukushima A, Iwasa M, Nakabayashi R, Kobayashi M, Nishizawa T, Okazaki Y, Saito K, Kusano M.
Reference Front Plant Sci. 2017 Aug 28;8:1464.

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Sample Information

Title Arabidopsis ecotype Columbia (Col-0)
Organism - Scientific Name Arabidopsis thaliana
Organism - ID NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation Arabidopsis ecotype Columbia (Col-0) was used. The mutant cad2-1 (Cobbett et al., 1998) was used as an allelic mutant of pad2-1 (Parisy et al., 2007). Further, 35S::GSH1 transgenic plants, 7-5 and 13-6 (Cheng et al., 2015), were also used to evaluate the shoot phenotypic changes. Plants other than those exposed to MV stress and P-lim were grown in Murashige and Skoog agar medium. Sterilized seeds were stratified at 5°C for 2 days and then sown on Murashige and Skoog medium containing 1% sucrose. Oxidative stress was produced by adding 0.05 μM MV to the Murashige and Skoog medium. The low phosphorus condition was created using P-lim medium in which the phosphate concentration was 20% of that in the Murashige and Skoog medium. Seedlings of Arabidopsis Col-0 and mutants were cultivated in growth chambers at 22°C under 16-h light/8-h dark conditions for 18 days (light strength, 80 μmol⋅m-2⋅s-1 of the photosynthetic photon flux). GSH1-overexpression lines were cultivated under the same condition for 20 days.
Sample Preparation Details ID SS1

Sample Preparation Details Information

Title Growth conditions
Description Plants were grown in MS agar medium except for the oxidative and phosphorous limited condition. Sterilized seeds were stratified at 5°C for 2 days, and were sown on Murashige and Skoog (MS) medium containing 1% sucrose (Control). Oxidative and Phosphorous limited conditions were produced adding 0.05μM Methyl Viologen and reducing phosphate concentration to 1/5 of MS medium. Seedlings of Arabidopsis Col-0 and the mutants were cultivated in growth chambers at 22°C in the 16-h light and 8-h dark condition for 18 days (light strength, 80 μmol m-2 s-1 of photosynthetic photon flux (PPF)). We sampled 20 independent plants (n = 20, biological replicates) for measurement of shoot biomass (flesh weight), 8 for metabolite profiling, and 3 for absolute glutathione quantification as follows.

Analytical Method Information

Method Details ID MS1
Sample Amount 1 μl of each sample (equivalent to 1.4 µg DW)

Analytical Method Details Information

Instrument GC Agilent 6890N gas chromatograph / MS Pegasus IV TOF mass spectrometer
Instrument Type
Ionization EI
Ion Mode positive
Description Extraction and derivatization for GC-TOF-MS

Each frozen sample with a 5-mm zirconia bead was extracted with 40 fold amount of solvent (methanol/chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds at 4°C in a mixer mill (MM301; Retsch, Haan, Germany) at a frequency of 15 Hz. Each isotope compound was adjusted to a final concentration of 15 ngper 1-μl injection volume. After 5-min centrifugation at 15,100 × g, a 200-μl aliquot of the supernatant was transferred to a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator (Thermo Fisher, Scientific, Waltham, MA, USA). We used extracts from 5-mg FW samples for derivatization, i.e., methoxymation and silylation. For methoxymation, 30 μl of methoxyamine hydrochloride (20 mg/ml in pyridine) were added to the sample. After 17 h of derivatization at room temperature the sample was trimethylsilylated for 1 h using 30 µl of MSTFA at 37°C with shaking. All derivatization steps were performed in a vacuum glove box VSC-100 (Sanplatec, Osaka, Japan) filled with 99.9995% (G3 grade) dry nitrogen.

GC-TOF-MS conditions
Using the splitless mode of a CTC CombiPALautosampler (CTC Analytics, Zwingen, Switzerland), 1 μl of each sample (equivalent to 1.4 µg DW) was injected into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, DE, USA) featuring a 30 m × 0.25 mm inner diameter fused-silica capillary column and a chemically bound 0.25-μl film Rxi-5 Sil MS stationary phase (RESTEK, Bellefonte, PA, USA) with a tandem connection to a fused silica tube (1 m, 0.15 mm). An MS column change interface (msNoVent-J; SGE, Yokohama, Japan) was used to prevent air and water from entering the MS during column change-over. Helium was the carrier gas at a constant flow rate of 1 ml min-1. The temperature program for GC-MS analysis started with a 2-min isothermal step at 80°C followed by 30°C temperature-ramping to a final temperature of 320°C that 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 222 sec. Data acquisition was on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA); the acquisition rate was 30 spectras-1 in the mass range of a mass-to-charge ratio of m/z = 60–800. Alkane standard mixtures (C8 - C20 and C21 - C40) purchased from Sigma-Aldrich (Tokyo, Japan) were used for calculating the retention index (RI) (Schauer et al., 2005). For quality control we injected methylstearate into every 6th sample. The sample run order was randomized in single-sequence analyses. We analyzed the standard compound mixtures using the same sequence analysis procedures.


Data Analysis Information

Title Data processing for GC-TOF-MS data
Data Analysis Details ID DS1
Recommended decimal places of m/z

Data Analysis Details Information

Title Data processing for GC-TOF-MS data
Description Nonprocessed MS data from GC-TOF-MS analysis were exported in NetCDF format generated by chromatography processing- and mass spectral deconvolution software (LecoChromaTOF version 3.22; LECO, St. Joseph, MI, USA) to MATLAB 6.5 or MATLAB2011b (Mathworks, Natick, MA, USA) for the performance of all data-pretreatment procedures, e.g. smoothing, alignment, time-window setting H-MCR, and RDA (Jonsson et al., 2006). The resolved MS spectra were matched against reference mass spectra using the NIST mass spectral search program for the NIST/EPA/NIH mass spectral library (version 2.0) and our custom software for peak-annotation written in JAVA. Peaks were identified or annotated based on their RIs, a comparison of the reference mass-spectra with the GolmMetabolome Database (GMD) released from CSB.DB (Kopka et al., 2005), and our in-house spectral library. The metabolites were identified by comparison with RIs from the library databases (GMD and our own library) and the RIs of authentic standards. The metabolites were defined as annotated metabolites after comparison with the mass spectra and the RIs from these two libraries. The data matrix was normalized using the CCMN algorithm for further analysis (Redestig et al., 2009) .
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