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Control plants were watered every day and sampled at the same time as drought-stressed plants. After sampling, six shoots or roots of each biological replicate were ground to a fine powder in liquid nitrogen http://www.selleck.cn/products/incb024360.html then pooled and stored at ?80��C prior to RNA purification, metabolite analysis, and chlorophyll analysis in the case of shoots. Total RNA was purified from roots and shoots separately, using TRIzol reagent (Invitrogen, http://www.invitrogen.com/). Probe labeling, chip hybridization, and scanning were performed according to the manufacturer��s instructions for the One Cycle Labeling Kit (Affymetrix, http://www.affymetrix.com/). Raw Affymetrix array data were normalized by robust multiarray averaging (RMA; Irizarry et?al., 2003). A present/absent call for each probe set was obtained using dChip (Li and Wong, 2001). Differentially expressed genes were selected based on the associative t-test (Dozmorov and Centola, 2003) using Matlab (MathWorks, http://www.mathworks.com/) with a Bonferroni-corrected P-value threshold of 8.16?��?10?7 (0.05/n, n?=?61278 probe sets), a P-value of http://www.selleckchem.com/products/bmn-673.html under accession number E-MEXP-3320. Metabolite extraction and analysis by GC-MS were performed according to Broeckling et?al. (2005). In brief, shoot and root samples were pulverized in liquid nitrogen and lyophilized under a partial vacuum at ?70��C. Six milligrams of dry tissue was extracted first with 1.5?ml of chloroform containing docosanol (a non-polar internal standard) then with 1.5?ml of HPLC-grade water containing ribitol (a polar internal standard). Following extraction, the mixture was centrifuged at 2900?g for 30?min at 4��C and 1?ml of each of the separated layers was collected, dried, and used in polar and non-polar metabolite analysis, respectively. Dried non-polar extracts were re-suspended in chloroform and hydrolyzed with HCl in methanol (MeOH) and then derivatized with N-methyl-N-trimethylsilytrifluoroacetamide w/1% trimethylchlorosilane (MSTFA + 1%TMCS; Pierce Biotechnology, http://www.piercenet.com/). Dried polar extracts were methoximated with methoxyamine-HCl http://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html in pyridine and then derivatized with MSTFA+1%TMCS. Both polar and non-polar samples were analyzed using an Agilent 6890 GC coupled to a 5973 MSD (http://www.chem.agilent.com/). The injection split ratio was 15:1 for polar samples and 1:1 for non-polar samples. Separation was achieved with a 60?m DB-5MS column (J&W Scientific, http://www.chem.agilent.com/). Metabolite analysis with UPLC-MS was done according to Brechenmacher et?al. (2010). Ten milligrams of pulverized and lyophilized sample (see above) was extracted with 80% methanol, containing 0.018?mg?ml?1 coumarin as an internal standard, by gentle agitation for 2?h.