5 d in saline conditions. The expression of the glutamate decarboxylases GAD3 and GAD4 showed a biphasic induction whereas the expression of GAD2 decreased at later timepoints. The GABA transporters GABA trans1 and GABA trans2 (AtGAT1) were also induced transcriptionally. We observed a rapid change in the ascorbate to dehydroascorbate ratio by salt stress. However, with the exception of a glutathione reductase, genes of the ascorbate/glutathione cycle did not respond at the transcriptional level. Taken together, metabolite levels and expression levels of genes encoding the corresponding metabolic enzymes showed co- regulation in some but not all of the pathways examined. The complex interrelation between gene expression and metabolite levels suggests that a multifaceted interplay at different control levels governs salt-induced metabolic re-adjustment. Correlation of the transcriptional and metabolic responses To compare changes in metabolite and transcript levels comprehensively, we generated a correlation matrix. The variance of metabolite and transcript amounts was adjusted to equal levels by z-transformation and a pairwise correlation was subsequently undertaken. The resulting matrix revealed two prominent clusters with opposite behaviours (Figure 3). The first cluster consisted of Metabolic Stress Adaptation metabolites including phenylalanine, maltose, serine, proline, raffinose and genes involved in metabolic regulation such as galactinol synthase (GolS), raffinose synthase (RS), pyrroline-5- carboxylate synthase (P5CS) and beta-amylase (BMY) which are strongly upregulated by high salinity. With respect to starch metabolism, the increase in maltose levels was positively correlated with the induction of beta-amylases (BMY7 and BMY8) at the transcriptional level but negatively correlated with the amount of starch. It is worth noting that starch showed the strongest negative correlation with raffinose. The negative correlated cluster also included phosphoric acid, shikimic acid and galactonic acid. In addition to the frequent association of metabolite levels with the expression levels of genes involved in the synthesis of these metabolites, a strong correlation between different metabolic pathways, e.g. starch mobilisation with raffinose and proline synthesis, was observed, suggesting a tight regulatory link between theses pathways in response to salt stress. Interestingly, the expression of ABA marker genes showed a strong co-regulation with numerous salt-induced metabolic changes. Transcript levels of RAB18, ABI2 and ABA-induced PP2C were negatively correlated with starch contents but showed a strong positive correlation with the levels of the starch degradation product maltose, suggesting that this part of the high salinity-induced metabolic reaction might be triggered by ABA. Figure 3. Correlation matrix of high salinity-induced metabolic and transcriptional changes. Pearson correlation coefficients of a pairwise correlation analysis of changes in metabolite levels and gene expression from all timepoints of two independent salt stress experiments. The background colours mark the value of the coefficient: orange: .0.91; yellow: .0.7; blue: ,20.7. Pearson correlation coefficients .0.91 and ,20.91 are significant (P-value ,0.05) and correlation coefficients .0.7 and ,20.7 are suggested to be biologically relevant. doi:10.1371/journal.pone.0003935.g003 PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3935