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. 2022 Aug 3;9:uhac171. doi: 10.1093/hr/uhac171

Figure 7.

Figure 7

Effects of MdPUB23 on MdICE1’s regulation of cold tolerance. (A) Cold-stress phenotypes of MdICE1-OX/MdPUB23-OX transgenic apple calli. WT, wild-type control; MdICE1-OX, MdICE1-overexpressing calli; MdICE1-OX/MdPUB23-OX, overexpression of MdPUB23 in the background of MdICE1-overexpressing calli; MdICE1-OX/MdPUB23-Anti, suppression of MdPUB23 in the background of MdICE1-overexpressing calli. Control, apple calli without cold treatment; Cold treatment, apple calli with cold treatment. Apple calli were grown on medium at 24°C for 5 d and then treated at 4°C for another 10 d. The experiments were repeated three times, and each experiment contained 8–10 calli subgroups per treatment. Bars = 1 cm. A representative picture is shown here. (B) The fresh weight of apple calli with or without cold-stress treatment shown in (A). Error bar denotes SD. Data are presented as mean ± SD of three independent experiments each containing 8–10 calli subgroups per treatment. Different letters above the bars indicate significant differences (p < 0.05) based on one-way ANOVA with Duncan's test. (C) DAB and (D) NBT staining of MdICE1 and MdPUB23 transient co-expressing apple leaves. Apple leaves were treated at 4°C for 1 d, and then, leaves were dyed with DAB and NBT. EV, the empty-vector control; MdICE1-OX, MdICE1-overexpressing apple leaves; MdICE1–OX/MdPUB23–OX, MdICE1 and MdPUB23 co-expressing apple leaves. Control, apple leaves without cold treatment; Cold treatment, apple leaves with cold treatment. The experiments were repeated three times with similar results, and each experiment contained 6–9 apple leaves per treatment. Bars = 1 cm. A representative picture is shown here. (E) Cold-stress phenotypes of MdICE1 or MdICE1–MdPUB23 transgenic Arabidopsis seedlings. Col-0, wild-type control; MdICE1-L1 and L2, MdICE1-overexpressing Arabidopsis seedlings; MdICE1-L1/MdPUB23-L1, MdICE1-L1/MdPUB23-L2, and MdICE1-L1/MdPUB23-L3, MdICE1 and MdPUB23 co-transformed Arabidopsis. Arabidopsis seedlings were grown on MS plates at 22°C for 8 d and then treated at −4°C for 1 h. The experiments were repeated three times, and each experiment contained 3–5 plates of Arabidopsis seedlings per treatment. A representative picture is shown here. (F) Survival rates of MdICE1 or MdICE1–MdPUB23 transgenic Arabidopsis seedlings shown in (E). Error bars denote standard deviations. Different letters above the bars indicate significant differences (p < 0.05) as obtained by one-way ANOVA tests.