Table 1.
Putative cis-elements in the promoter region of TaEXPA2 as predicted by PlantCARE.
Fig 1.
Expression profiles of TaEXPA2 in different tissues of wheat plants in response to abiotic stresses and signaling molecules, as detected by qRT-PCR.
(A) Expression of TaEXPA2 in different organs/tissues of wheat. (B-D) Total RNA was isolated from 2-week-old wheat seedling leaves that were collected after exposure to 20% PEG and 150 mM NaCl (B), 2 mM ABA, 50 mM GA (C), 10 mM MeJA, and 10 mM SA (D). Wheat seedlings incubated in sterile water (CK) were used as controls. The α-tubulin gene was used as an internal reference. The experiments were repeated at least three times.
Fig 2.
Subcellular localization of the TaEXPA2-GFP protein in onion epidermal cells.
The transient expression of 35S::GFP and 35S::TaEXPA2-GFP in onion epidermal cells. The cells were analyzed by laser confocal microscopy after culture on MS medium at 28°C for 2 days. Onion cell plasmolysis was induced by a 30% sucrose solution for 20 min before observation.
Fig 3.
Confirmation of TaEXPA2 transgenic tobacco plants.
(A) The relative expression level of TaEXPA2 was assessed by quantitative real-time PCR. The mRNA levels in the WT plants and five different TaEXPA2 transgenic lines (OE-6, OE-9, OE-15, OE-16, and OE-24). (B) TaEXPA2 protein abundance in the cell walls of WT and transgenic tobacco leaves by immunoblot analysis. Cell wall protein extracts were prepared from growing leaves of transgenic and WT tobacco plants. RuBisCO large subunit (Agrisera, AS03037) was used as a loading control. (C) Expansin activity in different transgenic lines and WT plants. Expansin activity was assayed by measuring the increase in the extension rate of wheat coleoptiles after the addition of the cell wall extract from leaves of different tobacco lines. Data represent the mean values for the three independent biological replicates. Standard errors are indicated by vertical bars. * and ** indicate significant differences from the WT values at P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.
Fig 4.
Seed production of WT and TaEXPA2 transgenic lines.
(A) Phenotypic comparison of WT and TaEXPA2 transgenic lines in the flowering stage. (B) The spike and capsules. (C) Total number of capsules per tobacco plant. (D) Distribution statistics of capsule length. (E) Seed yield of individual tobacco plants. (F) Total seed weight per tobacco plant. Data in (C), (D), and (F) represent the mean values for the six independent biological replicates. In (C) and (F), * and ** indicate significant differences from the WT values at P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.
Fig 5.
Seed size comparison and expression profile of TaEXPA2 during wheat-grain-filling in WT and transgenic plants.
(A) The level of TaEXPA2 expression during wheat-grain-filling by RT-PCR. The wheat grains were collected after pollination at intervals of 7 days to isolate total RNA. TaTubulin was used as an internal control. (B) Comparison of seed size in WT and TaEXPA2 transgenic tobacco plants. These images were obtained under the same magnification using a dissecting microscope (OLYMPUS SZX12). (C) Total weight of one thousand seeds. Data represent the mean values for the six independent biological replicates.
Fig 6.
Seed germination and young seedling development in TaEXPA2 transgenic lines and WT plants under water deficient stress.
(A) The germination of WT and TaEXPA2 transgenic tobacco seeds in the presence of 100 and 200 mM mannitol, and (B) the corresponding germination rate. The germination rate was counted 10 days after sowing. (C) Phenotypic analysis of 7-day-old WT and TaEXPA2 transgenic seedlings in the presence of 100 or 200 mM mannitol for 20 days. Primary root length (D) and lateral root numbers (E) were recorded. Each column represents an average of three replicates, and bars indicate SEs.
Fig 7.
Evaluation of the drought tolerance of TaEXPA2 transgenic tobacco plants.
(A) 10-day-old WT and transgenic plants were exposed to natural drought stress for 7 days and re-watered for 2 days, and (B) the corresponding survival rate of transgenic plants after drought stress and re-watering. (C) 50-day-old WT and transgenic plants were naturally exposed to drought for 21 days with the well-watered plants as controls. (D) The corresponding root growth status from (C). (E) Chlorophyll content (mg/g FW). (F) Net photosynthetic rate (Pn, μmol CO2 m-2 s-1). (G) Transpiration rate (E, mmol H2O m-2 s-1). (H) Leaf stomatal conductance (Gs, mmol H2O m-2 s-1). The experiment was replicated three times, and bars indicate SEs. * and ** indicate significant differences of P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.
Fig 8.
Evaluation of water loss, water potential, proline content, and kinetics of TaEXPA2 transgenic tobacco plants.
The corresponding relative water loss (A), water potential (B), and proline content (C) of the tobacco leaves undergoing water deficit stress shown in Fig 7. (D) The kinetics of water loss from the leaves of 50-day-old tobacco plants. Each column represents an average of three replicates, and bars indicate SEs. * and ** indicate significant differences at P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.
Fig 9.
Oxidation conditions in WT and TaEXPA2 transgenic plants under drought stress.
Changes in MDA content (A) and relative electrical conductivity (B) in WT and transgenic tobacco leaves under drought stress. (C) In situ detection of H2O2 and O2− by DAB staining (upper) and NBT staining (bottom) in WT and TaEXPA2 transgenic seedlings grown on normal medium for 14 days and then treated with 100 mM mannitol for 7 days. The experiment was replicated three times. (D) H2O2 content and (E) O2− production rate in 14-day-old plants treated with 100 mM mannitol for 7 days. Each column represents an average of three replicates, and bars indicate SEs. * and ** indicate significant differences of P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.
Fig 10.
The activity levels of antioxidant enzymes in tobacco plants under drought stress.
(A) Superoxide dismutase, SOD; (B) guaiacol peroxidase, POD; (C) catalase, CAT; (D) ascorbate peroxidase, APX; (E) monodehydroascorbate reductase, MDHAR; and (F) dehydroascorbate reductase, DHAR. Each column represents the mean ± standard error of three independent experiments. * and ** indicate significant differences from the WT values at of P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.
Fig 11.
The expression of antioxidant-related genes in WT and transgenic tobacco plants under drought stress, as detected by qPCR.
The transcript levels of these genes in transgenic plants are indicated relative to the levels in WT plants (taken as 1), and using transcripts of actin in the same samples as a reference. Each column represents the mean ± standard error of three replicates. * and ** indicate significant differences from the WT values at P < 0.05 and P < 0.01, respectively, according to Duncan’s multiple range test.