pathway to maltose [42–44]. Maltose was also found to accumulate in Lotus japonicus [38] and starch levels were reported to decrease in tomato leaves [45] upon prolonged salt stress, suggesting that starch mobilisation might be at the heart of the change in carbohydrate metabolism in response to saline conditions. In general, a stress-induced increase in maltose levels, a reduction in the amount of G6P and a decrease in the ratio of glucose to fructose may reflect an altered carbon flux upon salt stress. The dynamics of the overall changes in the metabolite composition induced by high soil salinity were confirmed in a second independent experiment (Figure 2A and Table S3). Transcriptional response of metabolism-related genes To address the question of whether the observed changes in metabolite levels correlate with the transcriptional response of genes encoding the corresponding metabolic enzymes, we explored the Arabidopsis transcriptome. We employed the MapMan gene ontology and image annotator [46,47] to examine the publicly available AtGeneExpress microarray data in order to identify metabolic genes that are significantly up- or downregu- lated by salt stress (Table S4). Numerous genes encoding isoforms of metabolic enzymes were found to be transcriptionally responsive to high salt stress. We focused our analyses on genes involved in starch metabolism, sugar conversion, the raffinose oligosaccharide (RFO) pathway and the ascorbate/glutathione cycle as the levels of metabolites involved in these pathways were significantly affected by salt treatment. AtGeneExpress microarray analyses were performed on young hydroponically-grown plants whereas metabolic profiles were obtained from soil-grown adult plants. To verify the transcrip- tional response in our experimental system and for direct comparison of the transcriptional and metabolic salt-induced patterns, we performed semi-quantitative RT-PCR on 48 selected genes from the same plant material which was used for metabolic profiling. 75% of the monitored genes displayed a similar Metabolic Stress Adaptation expression pattern upon high salinity treatment in young hydroponically-grown and in adult soil-grown plants (Table S4), emphasizing the robustness of the transcriptional response to high salt stress. Starch metabolism is very sensitive to the environment [48–55]. We observed a depletion of starch and an important increase in maltose levels upon exposure of plants to high salinity. Consistently, beta-amylase (BMY1 and BMY7), which cleaves maltose from starch-derived glucan molecules, was transcription- ally upregulated. Enhanced starch degradation by beta-amylase likely supplies energy and carbohydrate skeletons for adaptation processes (e.g. osmolyte production). Interestingly, genes involved in starch synthesis including an isoform of starch synthase and ADP-glucose pyrophosphorylase (AGPase1 and 2), which provides ADP-glucose, were also induced by high salinity. This observation might indicate that salt stress stimulates both starch mobilisation and starch synthesis. This interesting notion should be further addressed by metabolic flux analysis in future studies. High salinity induced alterations in the levels of soluble sugars (Table 1 and Figure 1B). Several genes encoding enzymes in the sugar conversion pathway were found to be upregulated by salt stress. The expression of genes encoding glucose-6-phosphate dehydrogenase (G6P DH), invertase (INV), hexokinase (HXK1 and 2) and UDP-glucose pyrophosphorylase (UGPase) were induced to different extents. In line with the accumulation of galactinol and raffinose, genes coding for galactinol synthase (GolS1, 2 and 3) and raffinose synthase (RS) were strongly induced by high salt conditions. Salt stress modified the expression of several genes encoding proteins involved in nitrogen metabolism. The majority of the monitored transcripts were strongly upregulated at later time- points including pyrroline-5-carboxylate synthase (P5CS1 and 2), phenyl ammonium lyase (PAL), nitrate reductase (Nry and NR) and proline oxidase (ERD5 and PrOX). Genes encoding different isoforms of glutamate decarboxylase (GAD) showed a diverse transcriptional response. GABA accumulated to high levels after Figure 2. Common and distinct metabolic responses to high salinity and ABA. The metabolic response of 4 week old soil-grown Arabidopsis plants to high soil salinity and ABA was compared. (A) Independent component analysis (ICA) of metabolite data after salt stress and ABA treatment. IC1 gives high loadings to maltose, proline, phenylalanine, glutamine and glycine. IC2 gives high loadings to raffinose, galactinol and G6P. (B) Schematic representation of the starch to sucrose and RFO pathways after 3 days of treatment. Metabolites (bold) and genes (italic) whose levels were upregulated in comparison to controls are indicated in red whereas blue represents downregulation. Orange and blue shading indicate up- and downregulation of the modules within the metabolic pathways, respectively. doi:10.1371/journal.pone.0003935.g002 PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e3935