Phenotypic analysis of growth patterns in wild-type seedlings and pp2c19 mutants. (a) Direction of hypocotyl growth in etiolated seedlings of wild-type (Col-0) seedlings and pp2c19 mutants on agar in PCR tubes under darkness (right panel). The images on the left show typical seedlings of wild-type seedlings and pp2c19-1 mutants on agar in PCR tubes. Scale bar = 5 mm. The central panel illustrates the degree of hypocotyl growth angle to the abaxial side (χ°) and the adaxial side (−χ°) of the hook. The box plots’ components are described in the legend of Fig. 1a. Asterisks indicate significant differences compared with the Col-0 (****P < .0001) as determined using one-way analysis of variance followed by Dunnett’s multiple comparison test. (b–f) Hypocotyl phototropism. Two-day-old etiolated seedlings grown on the agar inPCR tubes were mock preirradiated (b) or irradiated with overhead RL (RL: c–f) for 2 min at 20 µmol m^–2 s^–1 2 h before BL irradiation. BL irradiation of the hypocotyl was administered perpendicularly to the plane of the hook to induce a phototropic curvature, as described previously (Haga and Sakai 2012). (b) Pulse-induced, first positive phototropism without RL pretreatment. Etiolated seedlings were stimulated with unilateral BL for 1 min at the indicated fluence. Hypocotyl curvature was determined at 3 h after the onset of BL irradiation. Data shown are mean ± standard error (SE) from eight seedlings. (c) Pulse-induced, first positive phototropism with RL pretreatment. Etiolated seedlings were stimulated with unilateral BL for 1 min at the indicated fluence. Hypocotyl curvature was determined at 3 h after the onset of BL irradiation. Data shown are mean ± SE from eight seedlings. (d) Summary of fluence-response curves (Fig. 3b and c) of pulse-induced phototropism in wild-type (Col-0) seedlings and pp2c19-1 mutants. Red and black arrowheads show the peak of the fluence-response curves with or without RL pretreatment, respectively. (e) Time-course analysis of time-dependent phototropism. Following RL pretreatment, seedlings were stimulated with unilateral BL at 0.17 µmol m^–2 s^–1 for 27 min and then incubated under darkness. Hypocotyl curvature was determined at the indicated time points. Data shown are mean ± SE from 10 seedlings. (f) Time-course analysis of continuous light-induced phototropism. Following RL pretreatment, seedlings were stimulated with unilateral BL at 0.17 µmol m^–2 s^–1, and hypocotyl curvature was determined at the indicated time points. Data shown are mean ± SE from 10–12 seedlings. (g) Hypocotyl gravitropism. Following RL pretreatment, seedlings grown on the agar in PCR tubes were displaced horizontally under darkness. Gravistimulation was administered perpendicularly to the plane of the hook, and hypocotyl curvature of 23–24 seedlings was determined at 24 h after the onset of gravistimulation. The box plots’ components are described in the legend of Fig. 1a. (h) Time-course analysis of root phototropism. Two-day-old etiolated seedlings grown along the surface of vertically oriented agar medium were stimulated with unilateral BL at 100 µmol m^–2 s^–1, and root curvature was determined at the indicated time points. Data shown are means ± SE from 22 seedlings. (i) Elongation of hypocotyls. Two-day-old etiolated seedlings grown on the agar in PCR tubes were incubated under darkness for 8 h, and the growth increment of hypocotyls was determined by measuring the hypocotyl lengths of 22 seedlings. The box plots’ components are described in the legend of Fig. 1a. (j) Time-course analysis of root gravitropism. Two-day-old etiolated seedlings grown along the surface of vertically oriented agar medium were rotated at 90° under darkness, and root curvature was determined at the indicated time points. Data shown are mean ± SE from 14 seedlings. The differences between wild-type (Col-0) seedlings and pp2c19-1 mutants in Fig. 3g–j were not statistically significant (two-tailed Mann–Whitney U test, P < .05).