Fig 1.
Multiple sequence alignments of 10 DREBs belonging to the A-5 subgroup from different plant species.
The amino acid residues with 100% and more than 80% or 60% conservation among all sequences are indicated in white on a black or grey background or in black on a grey background, respectively. The AP2/ERF DNA-binding domain, NLS, and EAR motif are indicated. The asterisks indicate the 14th V and the 19th L residues inside the AP2/ERF domain. The accession numbers of all protein sequences are listed in S2 Table.
Fig 2.
Subcellular localization and transcriptional activation analysis of the AmDREB3 protein.
The pBI-AmDREB3:GFP fusion structure and control plasmid pBI-GFP were separately transferred into Arabidopsis leaf protoplasts using a polyethylene glycol-mediated method. (A) The images under dark, bright, and merged fields are presented. (B) Growth of the transformants with pGBKT7-AmDREB3, pGBKT7, or Y2HGold (the latter two were used as negative controls) on SD/-Trp or SD/-His medium and the blue color in the X-α-Gal assay are shown.
Fig 3.
Expression patterns of AmDREB3 under different abiotic stress conditions and in various organs.
One-month-old seedlings were subjected to different abiotic stressors or ABA treatment, and the expression changes of AmDREB3 in the shoots and roots are presented. (A) The expression changes during the 14 d drought treatment. The transcript level at 0 d was used as the control. (B) to (F) The expression changes during the salt (watering the seedlings with 350 mM NaCl solution), heat (42°C), cold (4°C for 24 h, 0°C for 12 h, and -6°C for 12 h, in total 48 h), UV-B (40 w), and ABA (100 μM) treatments for 2 to 48 h, respectively. (G) The expression pattern in different organs of naturally growing A. mongolicus shrubs in the wild. R, T, L, F, and P represent roots, twigs, leaves, flower buds, and immature pods, respectively. The α-tubulin gene of A. mongolicus was used as an internal reference gene. Data are presented as means ± SDs (n = 3). Asterisks and double asterisks indicate significant differences between each stress treatment or each organ and its control at p < 0.05 and <0.01, respectively.
Fig 4.
Constitutive expression of AmDREB3 enhanced osmotic stress tolerance of transgenic Arabidopsis at the germination stage.
(A) The location of transgenic lines and wild-type (WT) Arabidopsis on different medium plates. (B to D) After 10 d of germination on only 1/2 MS (0) and 1/2 MS plus 300 or 350 mM mannitol plates, respectively. (E and F) Germination rates and root lengths of seedlings on different media for 3 d and 10 d, respectively. Data are presented as means ± SDs (n = 3). Asterisks and double asterisks indicate significant differences of transgenic lines (OE-1, OE-4, OE-13, and OE-15) compared to wild-type (WT) at p < 0.05 and <0.01 levels, respectively.
Fig 5.
Constitutive expression of AmDREB3 enhanced drought tolerance of transgenic Arabidopsis at the seedling stage.
Ten-day-old seedlings were unwatered for 17 d and then re-watered. (A and B) Seedlings and plants before and 17 d after the suspended watering, respectively. (C and D) Plant phenotypes and survival rates after 7 d of re-watering. (E) Water loss of the detached shoots during a 7-h period. Shoots were weighed at 0.5 h intervals. Water loss is represented as the percentage of initial fresh weight (0 h) at each time point. Data are presented as means. Asterisk and double asterisks indicate significant differences of transgenic lines (OE-1, OE-13, and OE-15) compared to wild-type (WT) at p < 0.05 and <0.01, respectively.
Fig 6.
The changes in physiological indices in AmDREB3 transgenic Arabidopsis under drought stress.
Three-week-old seedlings were suspended watering for 10 d, and the contents of anthocyanins, H2O2, and MDA as well as the activities of CAT, POD, and SOD were measured before (control) and after the drought treatment, respectively. Data are presented as means ± SDs (n = 3). Asterisks and double asterisks indicate significant differences of transgenic lines (OE-1, OE-13, and OE-15) compared to wild-type (WT) at p < 0.05 and <0.01, respectively.
Fig 7.
Constitutive expression of AmDREB3 enhanced salt tolerance of transgenic Arabidopsis at the seedling stage.
Ten-day-old seedlings were watered with a 300 mM NaCl solution once and then were returned to their regular watering schedule of once every 5 d with tap water. (A, B and C) Plant phenotypes of the transgenic lines (OE-1, OE-13, and OE-15) and wild-type (WT) Arabidopsis before, 7 d after, and 14 d after watering with the NaCl solution, respectively. (D and E) Fresh weights and survival rates of plants after 7 d and 14 d of watering with the NaCl solution, respectively. Data are presented as means ± SDs (n = 3). Asterisks and double asterisks indicate significant differences of transgenic lines (OE-1, OE-13, and OE-15) compared to wild-type (WT) at p < 0.05 and <0.01, respectively.
Fig 8.
The changes in physiological indices in AmDREB3 transgenic Arabidopsis under salt stress.
Three-week-old seedlings were watered with a 300 mM NaCl solution, and the contents of anthocyanins, H2O2, and MDA as well as the activities of CAT, POD, and SOD were measured before (control) and 5 d after watering with the NaCl solution. Data are presented as means ± SDs (n = 3). Asterisks and double asterisks indicate significant differences of transgenic lines (OE-1, OE-13, and OE-15) compared to wild-type (WT) at p < 0.05 and <0.01, respectively.
Fig 9.
Constitutive expression of AmDREB3 enhanced heat stress tolerance of transgenic Arabidopsis.
Ten-day-old seedlings were placed at 53°C for 2 h after a pretreatment at 37°C for 1 h and at 24°C for an additional 2 h. Then the seedlings were returned to normal conditions for recovery. (A) Seedlings of transgenic lines (OE-1, OE-13, and OE-15) and wild-type (WT) Arabidopsis before the heat treatment. (B and C) Phenotypes of the plants at 3 d and 14 d after the heat treatment, respectively. (D and E) Fresh weights and plant heights after 14 d of recovery, respectively. Data are presented as means ± SDs (n = 3). Asterisks and double asterisks indicate significant differences of transgenic lines compared to WT at p < 0.05 and <0.01, respectively.
Fig 10.
Transcript levels of seven stress-inducible genes in AmDREB3 transgenic Arabidopsis.
The transcript levels of seven stress-inducible genes in two-week-old seedlings were examined by RT-qPCR analysis. The seedlings were exposed to osmotic stress (350 mM mannitol), salt stress (175 mM NaCl), or heat stress (37°C) for 5 h before sampling. Asterisks and double asterisks indicate significant differences of transgenic lines (OE-1 and OE-13) compared to wild-type (WT) at p < 0.05 and <0.01, respectively.
Table 1.
Predicted AmDREB3 promoter cis-acting elements.