Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress
(A) A subset of the crk mutants showed altered water loss (shown as decrease of fresh weight) compared to Col-0 wild type plants after detachment of shoots from roots as evaluated from rosette weight. Complementation of the crk2 (B), crk5 (C) or crk45 (D) mutants restored a wild type-like water loss phenotype as interpreted from decrease of fresh weight of excised rosettes. Asterisks indicate differences between crk mutants or complementation lines and Col-0 with statistical significance at *P<0.05, **P<0.01 and ***P<0.001 according to one-way ANOVA with post hoc Tukey HSD. The experiment was repeated three times with similar results. (E) Stomatal apertures were measured 2 h after abscisic acid (ABA) treatment. Some crk mutants are impaired in stomatal closure 2 h after treatment with 10 μM ABA. Results are means of % stomatal aperture ratio (width/length) ± SE (average number of stomata measured = 250). Asterisks indicate statistical significance between control and ABA treatment at *P<0.05, **P<0.01 and ***P<0.001 (linear model, single-step p-value adjustment). Lowercase letters indicate statistical significance between wild type Col-0 and crk mutant at P<0.05 (a), P<0.01 (b) and P<0.001 (c) according to one-way ANOVA with post hoc Dunnett’s test. (F) Stomatal density (number of stomata/mm2) is correlated with stomatal length (μm). Most of the crks exhibit a smaller stomata density which correlates with longer stomata (Pearson correlation -0.69, p-value = 0.04). Results are means (average number of stomata measured = 500). (G-I) Time courses of stomatal conductance (relative units) in response to a 3 min pulse of 500–600 ppb of O3 (G), darkness (H) and elevation of CO2 from 400 ppm to 800 ppm (I) in a subset of crk mutants and Col-0. Stimuli were applied at 0 time point, which is indicated by an arrow; pre-treatment stomatal conductance was used for normalization. Graph shows the mean of two experiments (n = 6). (J) Overexpression of CRK5 led to lower stomatal conductance compared to Col-0 wild type. (K) Complementation of the crk5 mutant restored wild type-like phenotype in the response to a 3-min pulse of 500–600 ppb of O3, darkness, and elevating CO2 from 400 to 800 ppm. Asterisks indicate differences between crk mutants or complementation lines and Col-0 with statistical significance at *P<0.05 according to one-way ANOVA with post hoc Tukey HSD. The experiment was repeated three times with similar results.