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	Figure 2. RWC and expression of TaAQP7 in wheat seedlings with different treatments.Ten-day-old wheat seedlings were subjected to 12 h dehydration treatment and the wheat leaves were sampled to measure RWC (A) and the expression of TaAQP7 (B). Ten-day-old wheat seedlings were treated with 20% of PEG6000 (C), 100 µM ABA (D), and 10 mM H2O2 (E) and leaves were sampled within 24 h to extract RNA for qRT-PCR analysis. The relative fold difference in mRNA level was calculated using the 2–ΔΔCt formula with TaActin as internal control. The mRNA fold difference was relative to that of distilled water treated samples used as calibrator. Vertical bars indicate ±SE of four replicates on one sample. Asterisks indicate significant difference between the RWC or relative fold difference in mRNA level (*p<0.05; **p<0.01). When no bar is shown, the deviation is smaller than the symbol. Three biological experiments were performed, which produced similar results.
	Figure 3. Effects of inhibitors of signaling molecules on the TaAQP7 transcripts under PEG and ABA treatments.A: Effects of pretreatment with inhibitors of ABA biosynthesis and scavenger H2O2 on the expression of TaAQP7 in the leaves of wheat seedlings exposed to PEG. The plants were pretreated with distilled water, 1 mM tungstate, and 5 mM DMTU for 2 h and 6 h respectively, and then exposed to 20% PEG6000 for 2 h and 6 h respectively. The treatment of tungstate or DMTU alone was also performed in the experiment. The plants treated with distilled water for 2 h or 6 h were used as control. B: Effects of pretreatment with scavenger of H2O2 on the expression of TaAQP7 in the leaves of wheat seedlings exposed to ABA. The plants were pretreated with distilled water, and 5 mM DMTU for 2 h and 6 h respectively, and then exposed to100 µM ABA for 2 h and 6 h respectively. The treatment of DMTU alone was also performed in the experiment. The plants treated with distilled water for 2 h or 6 h were used as control. The y-axis represents the relative fold difference in mRNA level calculated using the 2–ΔΔCt formula with TaActin as internal control. The mRNA fold difference was relative to that of distilled water treated samples used as calibrator. Vertical bars indicate ±SE of four replicates on one sample. Asterisks indicate significant difference between the expression of TaAQP7 under different treatment (*p<0.05; **p<0.01). When no bar is shown, the deviation is smaller than the symbol. Three biological experiments were performed, which produced similar results.
	Figure 4. Enhanced drought tolerance in transgenic lines when compared with the WT and VC.The WT, VC and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the plants were transplanted to containers filled with a mixture of soil and sand (3∶1) where they were regularly watered for two or five weeks. Six-week-old seedlings of transgenic and WT tobacco plants grown in pots were deprived of water for 20 d and the photos were taken (A). Three-week-old seedlings of transgenic and WT tobacco plants grown in pots were deprived of water for 20 d and the photos were taken (B). The whole two-week-old seedlings of transgenic plants and WT were used to extract RNA to detect TaAQP7 expression by RT-PCR with NtUbiquitin as an internal control (C). Three biological experiments were performed, which produced similar results.
	Figure 5. Osmotic tolerance analysis of TaAQP7-overexpressing plants.A total of 200 surface-sterilized seeds of each transgenic line, WT or VC germinated on MS medium containing 0 (A, a) or 300 mM (B, b) mannitol for 12 d, and the germination rate was calculated. A, B are photos of the first 12 days after germination on mediums. The WT, VC and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the seedlings were transplanted to MS or MS supplied with 150 or 300 mM mannitol for one week. The photographs were taken (C, D, E) and root length was calculated (F). Vertical bars indicate ±SE calculated from four replicates. Asterisks indicate significant difference between the WT and the three transgenic lines (*p<0.05; **p<0.01). Three biological experiments were performed, which produced similar results.
	Figure 6. Analysis of RWC, IL and MDA in transgenic lines under drought stress.The WT and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the plants were transplanted to containers filled with a mixture of soil and sand (3∶1) where they were regularly watered for two weeks. Three-week-old tobacco plants were deprived of water for 30 d. Tobacco leaves were sampled from WT and transgenic lines under drought stress for 15 d and 30 d to detect RWC (A), IL (B) and MDA (C). Vertical bars indicate ±SD calculated from four replicates. Asterisks indicate significant difference between the WT and the three transgenic lines (*p<0.05; **p<0.01). Three biological experiments were performed, which produced similar results.
	Figure 7. Analysis of H2O2 content, SOD and CAT activities in transgenic lines under drought stress.The WT and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the plants were transplanted to containers filled with a mixture of soil and sand (3∶1) where they were regularly watered for two weeks. Three-week-old tobacco plants were deprived of water for 30 d. Tobacco leaves were sampled from WT and transgenic lines under drought stress for 15 d and 30 d to detect H2O2 content (A), SOD (B) and CAT (C) activities. Vertical bars indicate ±SD calculated from four replicates. Asterisks indicate significant difference between the WT and the three transgenic lines (*p<0.05; **p<0.01). Three biological experiments were performed, which produced similar results.
	Figure 8. Analysis of H2O2 accumulation, SOD and CAT activities in transgenic lines under osmotic stress.The WT and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the seedlings were transplanted to MS or MS with 300 mM mannitol for one week. The whole seedlings were used to detect H2O2 accumulation, SOD and CAT activities. (A) In situ detection of H2O2 by DAB staining of WT and transgenic seedlings. (B) H2O2 content. (C) SOD activity. (D) CAT activity. Vertical bars indicate ±SD calculated from four replicates. Asterisks indicate significant difference between the WT and the three transgenic lines (*p<0.05; **p<0.01). Three biological experiments were performed, which produced similar results.
	Figure 9. The IL, MDA, H2O2 content and the activities of SOD and CAT in WT and transgenic lines with the same water status under drought conditions.The WT and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the plants were transplanted to containers filled with a mixture of soil and sand (3∶1) where they were regularly watered for two weeks. Three-week-old tobacco plants were deprived of water for 16 d. Tobacco leaves were sampled from WT and transgenic lines under drought stress to detect the RWC (A). When WT with 8 d drought treatment and transgenic lines with 16 d drought treatment, they maintained same water status and then the IL (B), MDA (C), H2O2 (D), SOD (E) and CAT (F) were measured.
	Figure 10. Subcellular localization of TaAQP7 protein.Onion epidermal cells transiently co-transformed with TaAQP7::GFP and pm-rk (Plasma membrane marker). (A, a) Fluorescence image of epidermal cell expressing the p35S-TaAQP7::GFP fusion protein. (B, b) Fluorescence image of epidermal cell expressing the pm-rk. (C, c) Merged fluorescence image of epidermal cell expressing the p35S-TaAQP7::GFP fusion protein and pm-rk marker. Images are dark field (A, B, C), bright field (a, b, c). Microscopy images of tobacco root cortical cells transformed with GFP alone as a control or TaAQP7. (D) Control; (E) OE6; (F) OE9; (G) OE13. Three biological experiments were performed, which produced similar results.
	Figure 1. Water channel activity test of TaAQP7.(A) The swelling rates of Xenopus laevis oocytes injected with cRNA encoding TaAQP7 or water (as negative control). The rate of oocyte swelling upon immersion in hypo-osmotic medium is plotted as V/V0 versus time, where V is the volume at a given time point and V0 is the initial volume. (B) Osmotic water permeability coefficient (Pf) of oocytes injected with cRNA encoding TaAQP7 or water. The Pf values were calculated from the rate of oocyte swelling. Vertical bars indicate ±SE of three replicates on one sample (Each replicate contains three oocytes). Asterisks indicate significant difference between oocytes injected with cRNA encoding TaAQP7 and water (*p<0.05; **p<0.01). Three biological experiments were performed, which produced similar results.

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