Distinct and common metabolic responses to high salinity and ABA To analyse the notion that ABA might be important for metabolic regulation and that ABA could be involved in carbon mobilisation, we studied the impact of ABA on metabolism. First, we examined the AtGeneExpress database for the transcriptional behaviour of salt-induced metabolic genes for their response to ABA. In agreement with the importance of ABA in the regulation of gene expression, the majority of the selected salinity-induced metabolic genes were also found to be upregulated by ABA (Table S4). We next investigated the dynamic response of the Arabidopsis metabolome to ABA in a time course experiment. Four week old soil-grown Arabidopsis plants were treated with 25 mM ABA for 2 h, 6 h, 24 h and 3 d. Subsequently, the metabolic profile was determined by GC-MS (Tables S5 and S6). Comparison of the metabolic profiles of NaCl- and ABA-treated plants in an independent component analysis (ICA) revealed common and divergent metabolic responses (Figure 2A and Table S7). The first independent component (IC1) gives high loadings to metabolites with a similar behaviour in salt- and ABA-treated plants. IC1 also reflects the time response. The second independent component (IC2) distinguishes metabolites which responded differently to salt and ABA treatment. The clear separation of metabolic reactions common to high salinity and ABA treatment from those which are distinct indicates the existence of ABA-dependent and indepen- dent metabolic responses within the high salinity pathway. Interestingly, IC2 gives a high ranking to raffinose, galactinol and G6P. Evaluation of the response of the starch to sucrose including the RFO pathway to high salinity or to ABA revealed common and stimulus-specific patterns (Figure 2B). Both high salt stress and ABA led to depletion of starch and a strong increase in maltose levels. Concordantly, beta-amylase, BMY7, is upregulated by both treatments. However, the next segment of the pathway responded in a manner opposite to that with salt or ABA. High salinity induced a significant decrease of the glucose to fructose ratio and a reduction of G6P levels whereas the glucose to fructose ratio (Table S2) and the G6P levels were upregulated by ABA. HXK1 and 2 were transcriptionally induced by high salt but not by ABA. The difference in the response to salt and ABA proceeded in the RFO pathway. While transcripts for galactinol synthase and raffinose synthase were upregulated upon both salt and ABA treatment, galactinol and raffinose accumulated under high salinity conditions but not in response to ABA. Altered carbon supply as well as post-transcriptional regulatory processes might lie at the base of these differences. The opposite behaviour of the glucose to fructose ratio and of G6P and F6P upon salt and ABA treatment indicates that a distinct carbohydrate flux might account for the deficiency of galactinol and raffinose accumulation upon ABA treatment. Consistent with our findings, other comprehensive studies have shown that changes in transcript levels are only partially reflected in concomitant changes in mRNA translation, enzyme activities or metabolite levels [56–58], indicating that environmental inputs impinge on different regulatory layers and highlight that multiple layers need to be analysed to understand the complex response of plants to stress. ABA is a central hormonal signal that regulates several aspects of stress response. With regard to metabolic adjustment to high saline conditions, ABA appears to trigger stress-induced starch mobilisation whereas salt-specific signals might be necessary for a complete metabolic adjustment to high salt. The correlation of various metabolites and genes involved in metabolic regulation Metabolic Stress Adaptation with plants’ sodium levels (Figure 3) suggests that the ion component of salt stress is involved in eliciting a part of the salinity-induced metabolic rearrangement. Since ABA is a key component involved in mediating different environmental stress conditions and plants respond to different stresses with overlapping but yet distinct physiological mechanisms, it is reasonable to assume that ABA-dependent signalling pathways act in concert with ABA-independent, stress-specific pathways to induce the appropriate intrinsic metabolic response. Taken together, non-targeted and hypothesis-driven experi- ments were combined to identify regulatory components of salt stress signalling at the metabolite level. In this way, we discovered ABA-induced and ABA-independent steps of salt stress-induced metabolic rearrangement. A differential regulation of carbon flux or utilisation was identified upon salt and ABA treatment. Interestingly, as depicted with the RFO pathway, the expression of salt-responsive metabolic genes mainly depends on ABA while ABA only triggers a partial metabolic reaction. The presented data indicate that starch mobilisation is a fast reaction common to both high salinity and ABA-treatment. As stress-induced starch exhaustion impairs growth, identification of the ABA-responsive- ness of the starch breakdown machinery now enables the identification of novel regulatory components/mechanisms and opens up new perspectives for improving yield and biomass production under unfavourable environmental conditions. Materials and Methods Plant material, plant growth and treatments Soil grown Arabidopsis thaliana Col-0 plants were cultivated in an 8 h light to 16 h dark regime at 22uC, 150 mE light intensity and 60% humidity. High salinity stress was applied by watering soil- grown plants with 150 mM sodium chloride. ABA is synthesised in roots and leaves. Thus, plants were sprayed and watered with a 25 mM (+)-ABA (Sigma) aqueous solution. For prolonged ABA treatment, plants were watered and sprayed every day. For both treatments, all plants were well-turgoured and stayed green during the entire experimental period. Ion measurements Soluble sodium and potassium concentrations were measured by flame photometry (atom emission spectroscopy (AES)). For this, 50 mg of pulverised plant material was extracted with 20 ml of double distilled water and the supernatant was used for analysis. Metabolite profiling and analysis of starch contents Ten or fifteen rosettes of 4 week old Arabidopsis plants were pooled and frozen in liquid nitrogen. 60 mg of ground plant material was extracted and analysed as described in [48]. Internal standards for quantitative and retention time index calibration were added as described in [59]. 160 ml of extract was dried and stored at –80uC until metabolite analysis. The solid fraction of the extract was used for starch measurements. For this, the organic phase was removed and the pellet washed twice with 80% ethanol. The pellet was incubated with 460 ml of 0.2 M KOH at 85uC for 1 h and subsequently the pH was adjusted with 140 ml of 1 M acetic acid to pH5. 100 ml of the supernatant was incubated over night with 1 U a-amylase, 1 U b-amylase and 1 U amylogluco- sidase. Free glucose was quantified using the glucose trinder kit (Sigma). Samples for metabolite profiling were prepared as described in [48]. Derivatised extracts of salt experiment 1 were analysed with GC-TOF-MS (Pegasus III; Leco) and chromatograms were processed with ChromaTof software. Metabolites of the second PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e3935