Figure 1.
Gene expression analysis and sub-cellular localization of OsGSTU4.
(A) Real-time PCR analysis showing transcript levels of OsGSTU4 in various tissues/developmental stages (ML, mature leaf; YL, Y leaf; P1-P6 stages of panicle development; S1–S5, stages of seed development) and under hormone (IAA, auxin; ABA, abscisic acid), abiotic stress (DS, dehydration; SS, salinity; OS, osmotic; CS, cold_ and oxidative stress (MV, methyl viologen; H2O2, hydrogen peroxide) treatments. The relative mRNA levels as compared to seedling (for tissues/developmental stages) and control mock-treated samples (hormone and stress treatments) have been presented. (B) The psGFPcs::GSTU4 construct used for transient expression in onion epidermal cells under the control of CaMV 35S promoter. (C) Images were observed in GFP filter (left), after DAPI staining (middle) and merged (right) by confocal microscopy after 24 h of incubation. In psGFPcs:OsGSTU4, GFP signal was detected in the nucleus and cytoplasm, whereas for psGFPcs (empty vector), GFP signal was detected in the whole cell. Arrows represent location of the nuclei.
Figure 2.
Expression of OsGSTU4 in E. coli and evaluation of stress responses.
(A) SDS-PAGE (12%) analysis showing induction and purification of 6xHis:OsGSTU4 protein (Lane 1, OsGSTU4 transformed uninduced cells; lane 2, OsGSTU4 transformed cells induced with 1 mM IPTG; lane 3, 6x:His:OsGSTU4 purified (∼25.5 kDa) protein). (B, C) Growth and GST activity of E. coli cells under various stresses (mentioned below each bar). (B) Growth of E. coli cells into empty vector (pET) and pET28a:OsGSTU4 (GST) transformed cells under different stress treatment. (C) GST activity in crude soluble protein, extracted from pET28a (pET) and pET28a:OsGSTU4 (GST) transformed E. coli cells under different stress conditions. Percentage of growth and activity under stress conditions has been given relative to the percentage of growth under control condition. Data were obtained from three independent replicates and are means ± SE. Data points marked with asterisk (*P≤0.05 and **P≤0.01) indicate statistically significant differences.
Figure 3.
Over-expression of OsGSTU4 in transgenic Arabidopsis plants.
(A) Schematic representation of 35S:OsGSTU4 construct used for over-expression in Arabidopsis. The complete ORF of OsGSTU4 was cloned in pBI121 vector carrying 35S promoter (B) Real-time PCR analysis of WT and transgenic Arabidopsis plants, showing the expression of OsGSTU4 in five homozygous transgenic lines (19A, 21A, 25A, 26A and 28A) with no detectable expression in WT. The expression of OsGSTU4 in homozygous transgenic lines are shown relative to 26A line exhibiting the lowest expression of the transgene. The expression of PP2A gene was used as internal control. Data are mean ± SE from three biological replicates.
Figure 4.
Effect of auxin and stress hormone ABA on transgenic plants.
Reduced root growth inhibition (A, B) of 35S:OsGSTU4 transgenic lines in the presence of IAA (A) and ABA (B). Root length of transgenic Arabidopsis and WT seedlings were measured after 5 days of growth on MS medium or IAA and ABA supplemented MS medium. Root growth inhibition of IAA and ABA treated seedlings was expressed as percentage of control incubated on IAA- and ABA-free medium. The experiment were repeated at least three times and values presented are mean ± SE. Data points marked with asterisk (*P≤0.05 and **P≤0.01) indicate statistically significant difference between WT and transgenic lines.
Figure 5.
Root growth and seed germination assays showing response of WT and OsGSTU4 over-expression transgenic plants to salinity and oxidative stress.
Comparison of root growth (A) and seed germination (B) of WT and transgenic Arabidopsis lines under salinity and oxidative stresses. (A) Increased percentage of relative root growth in transgenic Arabidopsis seedlings in response to salinity (150 mM NaCl) and oxidative (1 μM MV and 4 mM H2O2) stress. Root growth of seedlings under stress conditions was expressed as percentage of controls incubated on MS medium. (B) Enhanced seed germination in Arabidopsis transgenic lines expressing OsGSTU4 in the presence of 150 mM NaCl, 1 μM MV and 5 mM H2O2. The number of germinated seeds was expressed as the percentage of total number (40–60) of seeds plated. All the experiments were repeated three times and values are mean ± SE. Significance analysis was done by Student's t-test (*P≤0.05, **P≤0.01).
Figure 6.
Effect of salinity and oxidative stresses on OsGSTU4 over-expression Arabidopsis transgenic lines.
Comparison of chlorophyll content (A, B), plant height, biomass and survival rate (C, D) under salinity (A, C) and oxidative (B, D) stress conditions. (A, B) The chlorophyll content in WT and transgenic lines under stress condition are shown relative to the chlorophyll content under control condition. (C, D) Various phenotypic parameters of transgenic Arabidopsis and WT plants in the presence of 200 mM NaCl (C) and 25 μM MV (D) have been given. Percentage of growth (plant height), biomass and survival under salinity and oxidative stress condition was expressed as the percentage of plant growth under control condition. Values are mean ± SE from three independent experiments. Data points marked with asterisk (*P≤0.05) indicate statistically significant difference between WT and transgenic lines.
Figure 7.
GST activity and ROS levels of WT and transgenic plants during salinity and oxidative stresses.
GST activity (A), H2O2 (B) and O2•– (C) levels in transgenic Arabidopsis and WT plants under salinity and oxidative stress condition. (A) Graph showing increased GST activity in transgenic Arabidopsis lines expressing OsGSTU4 in the presence of 200 mM NaCl and 25 μM MV. (B, C) Decreased ROS [H2O2 (B) and O2•– (C)] level in the leaves of transgenic Arabidopsis as compared to WT in response to salinity and oxidative stress. The experiments were repeated at least three times and values are mean ± SE. Data points marked with asterisk (*P≤0.05) indicate statistically significant differences between WT and transgenic lines.
Figure 8.
Differential gene expression in over-expression transgenic lines.
(A) Significantly enriched GO biological process terms in the up-regulated genes. Node size is proportional to the number of transcripts in each category and colors shaded according to the significance level (white - no significant difference; color scale, yellow – P-value = 0.05, orange – P-value <0.0000005). (B) Heatmaps show the differential expression of genes involved in various biological processes and molecular functions found enriched in genes up-regulated in transgenic line. The color scale at the bottom represents the log transformed signal intensity. (C) Real-time PCR analysis showing differential expression of selected genes in transgenic lines under control, and stress conditions. The graph displays the fold change in mRNA levels in WT and transgenic line under control and stress (NaCl and MV) conditions as compared to the WT-control.