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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

Title LC-q-TOF-MS (to detect secondary metabolites)
Method Details ID MS2
Sample Amount 1 μl

Analytical Method Details Information

Title LC-q-TOF-MS (to detect secondary metabolites)
Instrument LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof
Instrument Type UPLC-QTOF-MS
Ionization ESI
Ion Mode Positive
Description Extraction for LC-q-TOF-MS to detect secondary metabolites

Each frozen sample was extracted with5 fold amount of solvent (methanol/water [8:2 v/v]) containing reference compounds (0.5 mg/L of lidocaine ([M+H]+, m/z 235.1804) and 10-camphorsulfonic acid ([M-H]-, m/z 231.0691) using a mixer mill MM301 (Retsch) at a frequency of 20 Hz for 5 min at 4°C. After centrifugation for 10 min at 15,000 × g, the supernatant was transferred into a 2 ml tube. Aliquot of the extracts was filtered using an Oasis® HLB μelusion plate (30 μm, Waters Co., Massachusetts, US). The extracts were transferred into a 2 ml tube.

LC-q-TOF-MS conditions to detect secondary metabolites
After preparation of the extracts, the sample extracts (1 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). The analytical conditions were as follows. LC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μm, length 2.1× 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,temperature, 40°C; MS detection: capillary voltage, +3.0 keV, cone voltage, 25.0 V, source temperature, 120°C, desolvation temperature, 450°C, cone gas flow, 50 l per h; desolvation gas flow, 800 l per h; collision energy, 6 V; mass range, m/z 100‒1500; scan duration, 0.1 sec; interscan delay, 0.014 sec; mode, centroid; polarity, positive; Lockspray (Leucineenkephalin): scan duration, 1.0 sec; interscan delay, 0.1 sec. The data were recorded using MassLynx version 4.1 software (Waters).


Data Analysis Information

Title Data processing for LC-q-TOF-MS data to detect secondary metabolites
Data Analysis Details ID DS2
Recommended decimal places of m/z

Data Analysis Details Information

Title Data processing for LC-q-TOF-MS data to detect secondary metabolites
Description The data matrix was aligned by MassLynx version 4.1 (Waters). The profiling data files were converted to the NetCDF format using the DataBridge function of the MassLynx software. The data matrices were processed using in-house Perl script for alignment and deisotope with the set of NetCDF data files.

For normalization, intensity values of remained peaks was divided by those of the lidocaine ([M+H]+, m/z 235.1804) and 10-camphorsulfonic acid ([M-H]-, m/z 231.0691) after cutoff of the low-intensity peaks (less than 500 counts).

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