SE182:/S1/M1/D1

Sample Set Information

ID	TSE1340
Title	Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes.
Description	BACKGROUND: 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets. RESULTS: In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds. CONCLUSION: Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.
Authors	Diaz, C., Kusano, M., Sulpice, R., Araki, M., Redestig, H., Saito, K., Stitt, M. and Shin, R.
Reference	BMC Syst Biol. 2011 Nov 21;5:192. doi: 10.1186/1752-0509-5-192.
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PubMed

Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Plants were grown on low salt media (LSM; 1.25 mM KNO3, 2 mM Ca(NO3)2, 0.75 mM MgSO4, 0.5 mM KH2PO4, 50 μM H3BO3, 10 μM MnCl, 2 μM ZnSO4, 1.5 μM CuSO4, 0.075 μM NH4Mo7O24, 74 μM Fe-EDTA, pH 5.7) with 1% sucrose and 0.6% Seakem agarose at 22°C with 16 h daylight at 150 μmol m-2 s-1. The all Arabidopsis plants used in this study have the same ecotype background, Col-0. Plants overexpressing 14-3-3 kappa, 14-3-3 chi and 14-3-3 psi and the knockout mutants of 14-3-3 genes were used as described. For metabolomic profiling and enzyme activity analysis, three days after germination, plants were transferred onto new LSM plates and grown vertically. To reduce the effect by the position of plates in the growth chamber, plates were moved every two days. After two weeks, shoots and roots were harvested separately.
Sample Preparation Details ID
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Analytical Method Information

ID	M1
Title	GC-TOF-MS
Method Details ID	MS1
Sample Amount	1 μL
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Analytical Method Details Information

ID	MS1
Title	GC-TOF-MS
Instrument	GC Agilent 6890N gas chromatograph / MS Pegasus III TOF mass spectrometer
Instrument Type
Ionization	EI
Ion Mode	positive
Description	Metabolite profiling using GC-TOF-MS was performed as described previously. Briefly, three of the harvested shoot or root samples were pooled as a replicate. Six replicates per line were used for metabolite profiling. A total of 5 mg fresh weight of the shoot and root samples were subjected to derivatization. An equivalent 6 μg of the derivatized samples were injected into the GC-MS instrument. The non-processed data obtained were pre-processed using the hierarchical multivariate curve resolution method.
Comment_of_details

PubMed

Data Analysis Information

ID	D1
Title	Statistical analysis
Data Analysis Details ID	DS1
Recommended decimal places of m/z
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Data Analysis Details Information

ID	DS1
Title	Statistical analysis
Description	SIMCA-P +12 software (Umetrics, Umeå, Sweden) were used for multivariate statistical analyses (i.e., PCA and OPLS-DA) and the R statistical environment http://cran.r-project.org for other statistical analyses such as the cross-contribution compensating multiple standard normalization (CCMN) and calculation of a false discovery rate (FDR). The PCA and OPLS-DA models were used to visualize the high-dimensional data and determine the metabolomic variation between the control (wild type) and the mutants (ox and/or KO). PCA was carried out to show how different variables (metabolites) change in relation to each other. OPLS-DA, which is as an extension of the supervised multivariate regression method PLS, was employed to remove some variation which was uncorrelated to class separation. Outliers in the GC-MS data were identified using missing value robust PCA and removed prior to further analysis. Metabolite abundance estimates were log transformed and scaled to unit-variance where applicable. Analytical bias was monitored via 11 internal, isotope-labeled standards and removed using the CCMN. To validate OPLS-DA models, we applied analysis of variance of cross-validated predictive residuals (CV-ANOVA) in the SIMCA-P software. Differentially abundant metabolites were identified using the LIMMA package. Briefly, a linear model was fitted to each metabolite to compare the levels of wild type with levels in the mutants. Significant changes were declared for metabolites with a FDR level < 0.05. The day and night change of metabolites were analyzed as described. Three of the harvested shoots were pooled as a replicate and six to eight replicates per genotype were analyzed. For day condition, long-day-grown (16 h light/8 h dark) plants were harvested 1 hour before offset of light and for night condition the plants were harvested 1 hour before onset of light. All data sets were analyzed for statistical differences compared to wild type by t-test using Prism 5 program (GraphicPad software, La Jolla, USA).
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