Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress
(A and B) Treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) for two hours (A) or methyl viologen (MV) for 48 hours (B) resulted in stronger impairment of photosynthesis in a subset of crk mutants compared to Col-0 wild type as shown by changes in the maximum quantum yield of primary photochemistry (φPo). Significant differences in relation to wild type are indicated (n = 12, bars represent SD; one-way ANOVA with post hoc Tukey HSD) *** P<0.001. The experiment was repeated three times with similar results. (C) Clustering of DCMU and MV experiments. Red and blue indicate increased or decreased response with respect to Col-0, respectively (average of alleles). Color intensity is proportional to a Benjamini-Hochberg false discovery rate (FDR) adjusted Z statistic, which takes the estimated means and their variation into account. Roughly, |Z|>2 corresponds to an FDR<5%, and |Z|<2.6 to an FDR<1%. Results were clustered using a complete linkage algorithm with 1-Pearson correlation as distance. (D) In several crk mutant lines exposure to O3 resulted in elevated electrolyte leakage. Electrolyte leakage was measured at indicated time points after start of the O3 exposure that lasted 6 h (350 ppb). Mean values ± SD from two independent experiments are presented (n = 12, one-way ANOVA with post hoc Tukey HSD). (E) Xanthine-Xanthine Oxidase (X+XO) infiltration induced different levels of electrolyte leakage in crk mutants compared to wild type (0.1 U ml-1, 4 h). Mean values ± SD from three independent experiments are presented (n = 16, linear model with single-step p-value adjustment for multiple testing). (F) Treatment with ultraviolet-A (UV-A) and–B (UV-B) radiation led to increased electrolyte leakage in crks compared to wild type. Mean values ± SD from three independent experiments are presented (n = 12, one-way ANOVA with post hoc Tukey HSD). (G) Effect on NaCl on the germination crk seedlings at 6 days after stratification. Values represent mean of the ratio (germination percentage on 120 mM NaCl / percentage on control medium) for each line (n = 15, linear model, single-step p-value adjustment). Experiments were performed three times. Asterisks indicate statistically significant differences between crks and Col-0 (*P<0.05, **P<0.01 and ***P<0.001) (D-G). Error bars indicate ± SE (D, E and F). Relative electrolyte leakage was calculated as a ratio of the value measured at the indicated time and the total electrolyte leakage after freezing (D and E) or autoclaving the samples (F).