The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco

Abstract

Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13) showed significantly enhanced salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA) analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2-; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed) condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance.

Figure 1. Analyses of transgenic tobacco plants.

(a) Schematic map of plant expression gene construct pCAMBIA1301-SbMT-2. (b) Confirmation of transgenic lines by PCR amplification of uidA, hptII and SbMT-2 genes (Lane M is molecular weight markers, Lane PC is positive control of PCR using plasmid pCAMBIA1301-SbMT-2 as template, Lane WT is wild type control plant (non-transgenic) and Lane L is transgenic lines). (c) Over-expression of the SbMT-2 gene in transgenic lines compared to wild-type control plants (Lane WT is wild type control plant and Lane L is transgenic lines) analyzed by semi-quantitative Rt-PCR, where actin was used as internal gene control. (d) Histochemical GUS staining of WT and transgenic plants. (e) Southern blot analysis (Lane PC is positive control plasmid pCAMBIA1301-SbMT-2, Lane WT is wild type control plant (non-transgenic) and Lane L is transgenic lines).

Figure 2. Comparative morphology of WT and T₁ transgenic lines under different stress condition.

WT and four independent T₁ transgenic tobacco lines (L2, L4, L6 and L13) grown under control, salt (200 mM NaCl), osmotic (10% PEG), Zn (5 mM), Cu (0.2 mM) and Cd (0.2 mM) stress for 3 weeks.

Figure 3. Analysis of SbMT-2 transgenic tobacco lines under different stress condition.

Comparison of (a) shoot length (cm), (b) root length (cm), (c) fresh weight (mg) and (d) dry weight (mg) of wild type (WT) and T₁ transgenic lines (L2, L4, L6 and L13) grown under control, salt (200 mM NaCl), osmotic (10% PEG), Zn (5 mM), Cu (0.2 mM) and Cd (0.2 mM) stress for 3 weeks. Graph represents the mean ± SE (of three replicates; n = 3) followed by similar letters are significantly different according to Tukey HSD at P<0.05.

Figure 4. Leaf disc assay of transgenic tobacco lines (T₁) for different stress tolerance.

Leaf discs of WT, L2, L4, L6 and L13 transgenic lines respectively were floated in different stress solution for 8 days.

Figure 5. Analysis of SbMT-2 transgenic tobacco lines under different stress condition.

Comparison of (a) Chlorophyll content, (b) Relative water content, (c) Electrolyte leakage and (d) Membrane stability index of wild type (WT) and T₁ transgenic lines (L2, L4, L6 and L13) grown in hydroponic (control) and treated with salt (200 mM NaCl), osmotic (10% PEG), Zn (1 mM), Cu (0.5 mM) and Cd (0.5 mM) stress. Graph represents the mean ± SD (of three replicates; n = 3) followed by similar letters are significantly different according to Tukey HSD at P<0.05.

Figure 6. In vivo localization of O₂ ⁻ and H₂O₂ in WT and transgenic lines under different stress condition.

Comparison of (a) O₂ ⁻ and (b) H₂O₂ localization in wild type (WT) and T₁ transgenic lines (L2, L4, L6 and L13) grown in hydroponic (control) and treated with salt (200 mM NaCl), osmotic (10% PEG), Zn (1 mM), Cu (0.5 mM) and Cd (0.5 mM) stress.

Figure 7. Estimation of H₂O₂, proline and MDA in transgenic tobacco lines under different stress condition.

Comparison of (a) H₂O₂, (b) Proline and (c) MDA content of wild type (WT) and T₁ transgenic lines (L2, L4, L6 and L13) grown in hydroponic (control) and treated with salt (200 mM NaCl), osmotic (10% PEG), Zn (1 mM), Cu (0.5 mM) and Cd (0.5 mM) stress. Graph represents the mean ± SD (of three replicates; n = 3) followed by similar letters are significantly different according to Tukey HSD at P<0.05.

Figure 8. Metal ions content in transgenic tobacco lines under different stress condition.

Comparison of Zn⁺⁺, Cu⁺⁺ and Cd⁺⁺ content in WT and transgenic lines in root, stem and leaf under (a) control and (b) stress condition (1 mM Zn, 0.5 mM Cu and Cd). Graph represents the mean ± SD (of three replicates; n = 3) followed by similar letters are significantly different according to Tukey HSD at P<0.05.

Figure 9. Expression analysis of zinc transporter encoding genes in transgenic tobacco lines under different metal ion stress condition.

Comparison of relative fold expression of (a) NtZIP1 and (b) NtHMA-A gene under Zn (1 mM), Cu (0.5 mM) and Cd (0.5 mM) stress.

Figure 10. Expression analysis of antioxidant enzyme encoding genes in transgenic tobacco lines under different stress condition.

Comparison of relative fold expression of (a) NtSOD gene, (b) NtPOD gene and (c) NtAPX gene under salt (200 mM NaCl), osmotic (10% PEG), Zn (1mM), Cu (0.5 mM) and Cd (0.5 mM) stress.

Figure 11. A hypothetical model for the role of SbMT-2 in phyto-remediation and ROS modulation under abiotic stress.