FIGURE 9.

Freezing tolerance assays of ahk double mutants compared with the wild-type plants with or without cold acclimation. A, ahk2-2 ahk3-3 and ahk3-3 cre1-12 mutants survived after 4 h of freezing at −4 °C. Fourteen-day-old light-grown plants were treated for 4 h at −4 °C and photographed after 2 days of incubation at 23 °C for recovery. The percentage of the plants that survived was calculated. Experiments were conducted in triplicate, and the means ± S.E. were plotted. n ≥30. All mutants are derived from the Col-0 ecotype. Statistically significant changes compared with Col-0 plants were indicated by * when p < 0.05 or by ** when p < 0.01 (Student's t test), respectively. † denotes statistically significant changes with p < 0.05 among the ahk double mutants indicated. B, example plates showing plants subjected to freezing tolerance assays (A). One plate (inner diameter 150 × 20 mm) contains 10 plants per each control or mutant. C, treatment of the ahk2-2 ahk3-3 and ahk3-3 cre1-12 mutants cold-acclimated with freezing at −7 °C for 6 h. Fourteen-day-old light-grown seedlings were cold-acclimated for 3 days, treated at −7 °C for 6 h, and the plants that survived were counted after 2 days of incubation at 23 °C for recovery. The percentage of the plants that survived was calculated. Experiments were conducted in triplicate, and the means ± S.E. were plotted. n ≥30. Statistically significant changes with p < 0.01 (Student's t test) compared with the Col-0 plants are indicated by **. †† denotes statistically significant changes with p < 0.01 among ahk double mutants indicated. D, example plates showing plants subjected to freezing tolerance assays (C). One plate (inner diameter 150 × 20 mm) contains 10 plants per control or per mutant.