salt stress experiment and the ABA treatment were analysed using a GC-Quad/MS (MD 800; Thermo), and the data were processed with the Masslab software. For peak identification, a customised mass spectral and retention time index library of ,1000 non- redundant entries, which currently covers 360 identified metabolic components of plant, microbial and animal origin, was used [60]. Ascorbate measurements Ascorbate levels and the redox-state of the ascorbate pool were determined by reversed phase HPLC. 100 mg of frozen and ground plant material was extracted with 0.1 M HCl (1:5 = ma- terial:extraction buffer) under an argon safety atmosphere. Levels of free ascorbate were analysed directly. Total ascorbate levels were determined after a reduction step with 10 mM DTT. For this, the pH was adjusted with CHES buffer (250 mM, pH9.5). Subsequently, extracts were acidified and analysed by HPLC. Separation of ascorbate was performed using an ODS column (Aquasil C18; 250 mm64.6 mm i.d.; 5 mm; Thermo) and a Dionex HPLC system with UV detection at 254 nm. For separation, a multi-step gradient was used starting from 5% MeOH, 95% H2O (0–5 min) to 19% MeOH, 81% H2O (5– 12 min) and to 90% MeOH, 10% H2O (12–30 min hold time 5 min). The pH of the elution buffer was adjusted to 3.9 using 0.3 M acetic acid. Gene expression analysis Publicly available microarray data were generated in the frame of the AtGenExpress project (e.g. http://www.uni-tuebingen.de/ plantphys/AFGN/atgenex.htm). For high salinity stress, 18 day old Arabidopsis thaliana Col-0 plants were treated with 150 mM NaCl. A detailed protocol for the cultivation, stress exposure and sample harvesting was recently published [11]. For ABA treatment, 7 day old Arabidopsis thaliana Col-0 seedlings were treated with 10 mM ABA. Samples were taken after 30 min, 1 h and 3 h [61]. Cell files were downloaded from the NASC array database (http://affymetrix.arabidopsis.info/narrays/experiment- browse.pl) and processed using ROBIN freeware (http://www-de. mpimp-golm.mpg.de/profil/services/index.html). Expression changes of more than 4-fold were counted as relevant. Additional genes involved in the pathways of interest were included in the analyses. Transcript levels of selected genes were analysed by semi- quantitative RT-PCR. Oligonucleotide primers were designed based on gene models from the MIPS database (www.arabidopsis. com). The resulting amplicon length of the PCR products was about 120 bp. Primer sequences are listed in table S8. RT-PCR was performed from 2 mg total RNA, with oligo (dt) primers and M-MuLV Reverse Transcriptase (Q-BIOgene). PCR was performed using MBI Fermentas Taq Polymerase according to the manufacturer’s protocol. PCR products were separated on agarose gels stained with ethidium bromide. Transcript levels were determined by reading the pixel densities using Quantity-One 4.6.1 software (BIO RAD) and normalised to tubulin used as a constitutive control. Bioinformatics and statistical data analyses The statistical screening for significant metabolic changes among the monitored metabolites was performed using Microsoft Excel. PCA and ICA analysis [62,63] were performed using MetaGeneAlyse (http://metagenealyse.mpimp-golm.mpg.de/). The ICA algorithm applied is based on 5 components of a PCA and calculates the maximal independence of non-Gaussian distributed data. The directions of variance calculated by a PCA analysis in a multidimensional room are orthogonal to each other. The ICA calculates the directions of maximal independency of the Metabolic Stress Adaptation pre-analysed data set. PCA and ICA algorithms are unable to operate with missing values, thus all substances measured at all timepoints were included in the calculations. For comparison of gene expression with metabolite data, transcript and metabolite data were z-transformed prior to pairwise correlation (Pearson correlation coefficient) analysis. Supporting Information Figure S1 Soluble sodium and potassium levels in response to high soil salinity. Soluble sodium and potassium ion concentra- tions were determined from the same plant material that was used for metabolic profiling. 50 mg of pulverised plant material was extracted with 20 ml of double distilled water. The supernatant was measured by flame photometry (atom emission spectroscopy). Found at: doi:10.1371/journal.pone.0003935.s001 (0.01 MB PDF) Figure S2 Principal component analysis (PCA) of technical replicas of the salt stress time course experiment shown in Figure 1. To minimize missing data points, values from 3 out of 4 replicate samples were normalised to the average of 6 control samples and used for PCA. Found at: doi:10.1371/journal.pone.0003935.s002 (0.02 MB PDF) Table S1 Loadings of principal component analysis (PCA). Found at: doi:10.1371/journal.pone.0003935.s003 (0.05 PDF) MB Table S2 Glucose to fructose ratio. The relative glucose and fructose contents of plants treated for 3 d with NaCl or ABA were calculated from the data presented in Table 1 and Table S3. Bold letters indicate significant changes in metabolite levels (t-test; P- value ,0.05). Found at: doi:10.1371/journal.pone.0003935.s004 (0.01 MB PDF) Table S3 Dynamic changes in metabolite levels upon high soil salinity in an independent biological experiment. Four week old soil-grown Arabidopsis thaliana was watered with 150 mM NaCl. Metabolic profiles were determined from leaves of pools of 15 individual plants four times by GC-MS. Compounds were identified by the retention time (TAG RT), the quantitative mass tag (TAG MASS) and the reverse match value (REV MATCH) of the mass spectra comparison. Starch levels were quantified spectrophotometrically. Values are x-fold changes compared to the corresponding unstressed controls. Bold letters indicate significant changes in metabolite levels (t-test; P-value ,0.05). Found at: doi:10.1371/journal.pone.0003935.s005 (0.05 MB PDF) Table S4 Transcriptional response of metabolism-related genes to high salt and ABA treatment. Transcriptional response of genes encoding enzymes in selected metabolic pathways to NaCl and ABA treatment of young, hydroponically-grown seedlings (At- GeneExpress) and adult, soil-grown plants (RT-PCR, same plant material that was used for metabolic profiling). ++, strong transcriptional induction $7-fold for microarray and $2-fold for RT-PCR; +, transcriptional induction $4-fold for microarray and $1.5-fold for RT-PCR; o, unchanged; -, transcriptional reduction #0.5-fold; –, strong transcriptional reduction #0.15-fold; 2/+, transcriptional induction at late timepoints after transcriptional reduction at early timepoints; n.d., not determined. Found at: doi:10.1371/journal.pone.0003935.s006 (0.06 MB PDF) PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e3935