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Data processing<br />
Peak picking a … Data processing<br />
Peak picking and alignment: Raw CE-MS data were analyzed with the proprietary software MasterHands (Sugimoto et al., 2010b; Sugimoto et al., 2010c). In brief, peaks were detected from sliced electropherograms (0.02 m/z width), and the accurate m/z value for each peak was calculated by Gaussian curve fitting. Migration times of the detected peaks were normalized by a dynamic time-warping method; numerical parameters were optimized using the simplex method and matching peaks across multiple data sets by dynamic programming (Sugimoto et al., 2010a). Peaks were picked and aligned using this software.<br />
Peak annotation: Metabolites in the standard compounds were assigned to the remaining features by matching their m/z values and normalized migration times using the software described in Peak picking and alignment.<br />
Quantification: For normalization, the area of the detected peak was divided by the area of the internal standard peak. Based on the of calibration curves for standard compounds, metabolite amounts were quantified. In C. australis internodal cells, the vacuolar solution accounts for approximately 95% of the whole cell volume (Sakano and Tazawa, 1984). Therefore, in the present study, the cytoplasmic fraction was estimated to occupy about 5% of the total cell volume; this value was used to calculate metabolite levels in the cytoplasmic fractions.<br />
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Statistics<br />
Data transformation: Calculated amounts were standardized by subtracting the mean amount for each metabolite in a sequence experiment from the calculated amount of each sample and then dividing this value by the standard deviation of each metabolite (z-score). Levels of undetected metabolites were set at 0, and then standardized.<br />
Statistics: Standardized amounts were submitted for hierarchical clustering analysis (HCA) and processed with PermutMatrix software (http://www.atgc-montpellier.fr/permutmatrix) according to Euclidean distance and Ward’s method (Caraux and Pinloche, 2005; Ward, 1963).<br /> d Pinloche, 2005; Ward, 1963).<br />
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