Figure 1.
Expression pattern of OsGA2ox5 in vivo.
(A) Real-time PCR analysis of OsGA2ox5 in various organs of wild-type plants. RS, seedling root; CS, seedling culm; LS, seedling leaf; SS, seeding sheath; YP, young panical. The expression is relative to that of OsActin. Values are expressed as the average ± SD of three technical replicates, and the amount of OsGA2ox5 in roots was set at 1.0; (B) Histochemical analysis of POsGA2ox5:GUS gene activities in different tissues and organs of rice. The promoter region of OsGA2ox5, 3,500-bp upstream of ATG (POsGA2ox5) was inserted upstream of the GUS gene at the Xba I-Sma I sites of the p1300GN-GUS vector. Arrows indicated the expression tissues of OsGA2ox5.
Figure 2.
Localization of OsGA2ox5-YFP protein.
(A) Diagram of the inserted region of the vector pA7:: OsGA2ox5::YFP; (B) Subcellular localization of OsGA2ox5. OsGA2ox5 was detected both in the cytoplasm and nucleus, the nucleus marker protein OsGHD7 was detected exclusively in the nucleus of onion epidermal cells and the control YFP showing signal both in cytoplasm and nucleus. DIC (Differential Interference Contrast), referring to bright field images of the cells.
Figure 3.
Phenotype of OsGA2ox5-overexpressing plants.
(A) Phenotype of WT (left) and dwarf OsGA2ox5-ox plants (right). 2-week-old water cultured seedlings were used for photograph; (B) Arrows indicate the boundary between the second leaf sheath and the blade of 5-day-old water cultured seedlings. Bar = 1 cm; (C) Longitudinal sections of the elongated regions of the second leaf sheath of WT (left) and OsGA2ox5-ox plants (right). Bar = 25 µm; (D) Quantitative measurement of the cell length of second leaf sheath in WT and OX (n = 20). Error bars show standard errors (SE). Asterisks indicated significant differences at P <0.01 compared with the wild type by Student’s t test; (E) Ectopic expression of OsGA2ox5 in Arabidopsis. Left is OsGA2ox5 transgenic plants and wide type Arabidopsis (Col) is on the right. Plants photographed are 4-weeks-old. Bar = 2.5 cm; (F) Expression level of OsOsGA2ox5 in transgenic rice; WT was used as a control; (G) Southern blotting analysis of transgenic plants. Restriction endonuclease Hind III was used to digest the genomic DNA from the leaf tissue. M, molecular marker; WT, wild type; L7, L12, L13, three transgenic lines.
Figure 4.
Exogenous GA3 effectively reverses the GA-deficiency phenotype.
(A) Response to the application of GA3 in the plants. Two-week-old plants cultivated in MS liquid medium containing 1 µM GA3 or no GA3 for 1 week. Bar = 2 cm; (B) Plant elongation of OsGA2ox5-ox and WT seedlings treated with GA3. Plant height was measured at day 7 after GA3 treatment. Results represent three independent experiments with similar results. Error bars show standard errors (SE). Asterisks indicate significant difference at P <0.01 compared with the wild type by Student’s t test; (C) 1-month-old OsGA2ox5-ox and WT plants grown in a greenhouse and sprayed with exogenous GA3. Bar = 10 cm.
Figure 5.
The transcription levels of gibberellin metabolism and signal pathway genes expression levels analyzed by Real-time PCR. The rice OsActin gene was used as an internal control. Data are the mean ±SE of three independent measurements with three repeats. Asterisks indicate significant difference at P <0.01 compared with the wild type by Student’s t test. The expression of all of these GA biosynthesis and GA signaling genes was up-regulated in OX plants, especially the OsGA3ox1 gene nearly 10-fold up-regulated. 3-week-old rice plants cultivated in water were used for experiments. OsGA20ox1 and OsGA3ox1 encode GA biosynthesis enzymes; OsGA2ox1 encodes an enzyme that functions in GA degradation; OsSLR encodes a negative GA regulator in GA signaling and OsGID1 and OsGID2 encode GA receptors in rice.
Figure 6.
OsGA2ox5 affects root starch granule development and gravitropism.
(A) Starch staining and detection of wild-type and OsGA2ox5-ox plants root caps. i, Resin section of WT and OX plants root caps and ii, I2-KI staining of the WT and OX plants root caps. Figures showing that OX plants have increased cell layers in the enlarged root caps compared to WT; iii, starch content in root caps of WT and OX plants. Roots cap segments were excised about 1 cm and pooled into samples from 10 plants each for experiments. Results represent three independent experiments with similar results. Bar = 100µm. Asterisks indicate significant difference at P <0.01 compared with the wild type by Student’s t test; (B) Gravity response of light-grown 4-day-old WT and OX seedling roots. After reorientation the OX seedling roots bent faster than WT. θ indicated the roots bending angle of the WT and OX plants respectively after reorientation at 5h. Experiments were performed three times with similar results. Bar = 1 cm; (C) Time course of root gravitropical curvature (after reorientation). Light-grown wild-type and OsGA2ox5-ox seedlings were displaced by 90° and monitored for the orientation of the primary root caps. The vertical position is represented by 90°, and the horizontal position is represented by 0°. Data shown are the means ± SE of 30 seedlings; (D) Time course of root length (after reorientation). Data shown are the means ± SE of 20 seedlings.
Figure 7.
Effects of salinity stress on plant growth.
(A) Phenotype of WT and OX rice seedlings under salt treatment. Photographs were taken after 5 d of growth in water (control) and 100 mM or 140 mM NaCl. For each treatment, 20 seedlings were measured. Bar = 1 cm; (B) Statistics analysis of plants height under salt treatment. WT and OX plants stem height at 5 days of growth in various concentrations of NaCl. Bar = 1 cm. Asterisks indicate significant difference at P <0.01 compared with the wild type by Student’s t test; (C) Quantitative analysis of survival rates under salt treatment. The results are averages of three independent experiments with 30 plants per experiment. Asterisks indicate significant difference at P <0.01 compared with the wild type by Student’s t test; (D) Phenotype of wide type Arabidopsis (Col) and transgenic Arabidopsis with and without GA3 under salt treatment. Physiological changes in WT and OsGA2ox5-ox transgenic Arabidopsis plants transferred to plates containing 170 mM NaCl. Photographs were taken 3 weeks after transfer. +GA represents treatment with 10 µM GA3.