Ed under in a vial 55 mm high and 33 mm in diameter.
Ed under in a vial 55 mm high and 33 mm in diameter. 1.5 ml of distilled water was placed at the bottom of each vial, and the vials were closed and placed at 25 in darkness. After 24 h of wetting, the average percentage germination was determined. After50 dry caryopses, germinated seeds and dehydrated seeds per replicate were extracted for parallel metabolite profiling as described in [31]. Seeds were homogenized using previously cooled mortar and pestle with liquid nitrogen and extracted in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25679764 a pre-chilled methanol:chloroform:water extraction solution (1:2.5:1 v/v) for 30 min at 4 shaking. Standards, i.e. 0.2 mg/ml ribitol, 1 mg/ml ampicillin in water, 1 mg/ml corticosterone in methanol and 5 mg/ml heptadecanoic acid in chloroform, were subsequently added. After centrifugation at 2,200 g, the remaining pellet was extracted in a second step with 500 l methanol/chloroform. The extracts were combined and 500 l of water was added to the supernatant to order Ixazomib citrate separate the chloroform phase from the water/methanol phase. The latter was used for metabolite analysis via GC-MS DSQII (Thermo-Fisher ltd.) and UPLC-XevoQTOF-MSMS (Waters ltd) exactly as described in [29]. A volume of 200 l of water/methanol extract was reduced to dryness in vacuum. Residues were derivatized and analyzed via an established GC-MS based method adapted to seeds [32]. GC-MS data were processed by Xcalibur?and normalized by the internal standard ribitol. The UPLC raw data were recorded with the aid of MassLynx version 4.1 software (Waters ltd). Metabolites were identified by using MassLynx software and searched against metabolite database Chemspider (http://www.chemspider.com/). The quantification of the compounds is based on the relative peak response area of each mass signal after pareto scaling in the chromatograms and normalized to the tissue DW.Statistical analysisThe significance between the germination percentage of the caryopses and percentage of seeds that survived was tested by one-way ANOVA following arcsin transformation. Principal component analysis (PCA), t-testBai et al. BMC Plant Biology (2015) 15:Page 3 ofand ANOVA were implemented using the software TMEV [33]. The term significant is used in the text for p-values lower than 0.05 (p < 0.05).Network analysisThe coordinated behavior of metabolites can be delineated using graph theory, where the nodes represent metabolites and the relationship between them is demonstrated as edges. The generation of the graphs was based on the correlation analysis of all metabolites and the two physiological traits (germination and survival percentage). Prior to correlation analysis, each metabolite was normalized by its respective mean calculated across the time-point measurements. Physiological traits were arcsin transformed. In addition each component (metabolites and physiological traits) of the dataset was pareto-scaled. Normal distribution was tested across all time-points by employing a Shapiro-Wilk test. In most cases (dry seed network = 74.0 , germination network = 92.2 , dehydration network = 79.2 ) the assumption of normal distribution was violated. Thus, the non-parametric Spearman's rank correlation was chosen over the parametric Pearson correlation to compute correlation coefficients. To reconstruct a network capturing coordinated changes in metabolic and physiology profiles, first the corresponding p-value threshold Spearman rank correlation coefficient ensuring a q-value of 0.05 was determined. Second, the adequate.