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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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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.


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