Figure 7.

Relative changes of leaf primary metabolites in the nonacclimated and the 10°C cold-acclimating reil1-1 reil2-1 mutant compared with the Col-0 wild type. A, Heat map of relative changes compared with nonacclimated Col-0 (*). Mean ratios were log₂ transformed and color coded according to the scale at top (n = 6). Metabolites presented in C are indicated (x). Metabolites were arranged by hierarchical clustering using Euclidian distance and complete linkage. B, Principal component analysis of the data set shown in A. Col-0 in the nonacclimated state is indicated by an asterisk within the 0.00-h circle to the left. The gray arrow indicates the progressing metabolic changes of Col-0 in the course of cold acclimation. The black arrow indicates nonacclimated reil1-1 reil2-1. Time after the cold shift is coded by circle size. The 4-d annotation indicates the time point of reil1-1 reil2-1 divergence from the wild type. C, Comparative time course of selected metabolite pools normalized to nonacclimated Col-0 indicated by an asterisk within the 0.00-h circle to the left (means ± se; n = 6). Plants were precultivated at 20°C and shifted to 10°C conditions at developmental stage ∼1.10 according to scheme C in Figure 1. Samples were harvested immediately before (0.00 h) and following the cold shift. Cold shift and sampling time points at full days (d) or weeks (w), except samplings at 0.25 to 8 h, were at 6 h (±5 min) after dawn of a 14-h/10-h day/night cycle. Two independent experiments with three replicate samples each per time point were performed. Samples were pools of mature leaves from at least two plants. Metabolism was profiled by multitargeted gas chromatography-mass spectrometry (GC-MS)-based technology. Metabolites with significant changes compared with the wild type, changes in the course of cold acclimation, and significant interactions of both effects were selected by two-way ANOVA (P < 0.001; Supplemental Table S1).