Here, we investigated the metabolic response in comparison to the transcriptional reaction during the initial stages of salt stress in a time-dependent manner. Channelling of data from this compre- hensive approach to targeted analyses allowed us to unravel the impact of the phytohormone ABA on metabolic regulation. Results and Discussion Metabolic dynamics in response to high salt conditions Plant metabolism is highly flexible and has the potential to adjust to changing environmental conditions. To explore the temporal dynamics of the Arabidopsis thaliana metabolome in response to high soil salinity on a large scale, we performed metabolic profiling on leaves of plants exposed to high salt stress. Four week old soil-grown plants were watered with 150 mM NaCl and analysed 6 h, 12 h, 1 d, 3 d and 5 d after the onset of stress. While prolonged stress exposure led to sodium accumulation in leaves, the concentration of soluble sodium did not increase during the early experimental timepoints (Figure S1), indicating that the immediate metabolic modifications are not caused by ion toxicity. Comprehensive metabolic profiling was performed by gas chromatography-mass spectrometry (GC-MS) for polar com- pounds. Additionally, we determined the levels of starch, the main carbohydrate store in plants, and of ascorbate and dehydroascorbate, as parameters of cellular redox-balance. Principal component analysis (PCA) of the metabolic data revealed a continuous change of the metabolome upon high salt stress. We observed minor overall changes in metabolite levels early after application of high salt conditions but a pronounced alteration after prolonged stress (Figures 1A and S2 and Table 1 and S1) which is consistent with a recent analysis of in vitro-cultured Arabidopsis cells [29]. The clear separation of the 3 d and 5 d- metabolic profiles from the metabolome of unstressed plants highlights the plasticity of Arabidopsis’ metabolism upon salt stress. Analysis of individual metabolite levels showed that high salinity had a profound effect on central metabolism and induced complex changes in the metabolome in a time-dependent manner (Table 1 and Figure 1B). Temporal resolution revealed sustained and transient, early and late-responsive metabolic alterations. The Metabolic Stress Adaptation early response to high salinity (6 to 12 h) was characterised by an increase in raffinose, trehalose, tyrosine, serine, phenylalanine, glutamic acid, proline, gluconic acid and dehydroascorbate, and a decrease in maltose, fructose, glucose-6-phosphate (G6P) and ascorbic acid levels. Prolonged stress (3 and 5 d) induced a strong accumulation of raffinose. Moreover, the levels of maltose, galactinol, proline and c-amino butyric acid (GABA) were highly induced. Raffinose, galactinol and proline function as osmopro- tectants and accumulate under stress conditions [7,30–34]. GABA metabolism plays a major role in carbon and nitrogen metabolism. The strong increase in the amounts of GABA as a result of high salinity is consistent with its putative role in stress defence [35–37]. Upon long-lasting stress, the levels of amino acids including valine, tyrosine, serine, phenyalanine, glycine, glutamine and glutamic acid showed a coordinated increase which might be due to enhanced protein catabolism and/or re-allocation of nitrogen caused by growth inhibition. A similar accumulation of amino acids has been observed recently in Lotus japonicus and Populus euphratica, indicating a conservation of this response to high salinity [38,39]. Redox balance is crucial for cellular function. Application of high salt stress did not change the overall pool size of the antioxidant ascorbate. However, a rapid and sustained increase in oxidised dehydroascorbate (Dha) and a decrease in reduced ascorbate (Asc), which led to a marked shift in the Asc to Dha ratio, was observed (Figure 1B). These data indicate that high salinity has an immediate early impact on the cellular redox state. Interestingly, the levels of G6P, which is a source of the redox equivalent NADPH under stress conditions [40], decreased in parallel with Asc. High salt conditions induced a rapid alteration of carbohydrate levels. After irrigation of plants with NaCl, the levels of several soluble sugars and the glucose to fructose ratio changed significantly, indicating an impairment of central carbohydrate metabolism (Table S2). Notably, prolonged salt stress led to a substantial decrease in starch content and a strong increase in maltose (Table 1 and Figure 1B). Starch is the major carbohydrate store in plants and is important for energy and carbon skeleton supply [41]. In leaves, starch is degraded via the b-amylolytic Figure 1. Temporal dynamics of the Arabidopsis metabolome upon salt stress. Metabolic profiling was performed on control plants and on plants exposed to high soil salinity for 6 h, 12 h, 24 h, 3 d and 5 d. (A) Principal component analysis (PCA) on the metabolic dynamics. PC1 gives a high weighting to raffinose, GABA, galactinol and proline; PC2 to glutamine, glutamic acid and G6P. (B) Relative metabolite levels of selected metabolites upon salt stress. Relative metabolite levels were log2 transformed and standard deviations were calculated from four independent measurements of pools consisting of ten plants each. doi:10.1371/journal.pone.0003935.g001 PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3935