A novel gene SbSI-2 encoding nuclear protein from a halophyte confers abiotic stress tolerance in E. coli and tobacco

Abstract

Salicornia brachiata is an extreme halophyte that grows luxuriantly in coastal marshes. Previously, we have reported isolation and characterization of ESTs from Salicornia with large number of novel/unknown salt-responsive gene sequences. In this study, we have selected a novel salt-inducible gene SbSI-2 (Salicornia brachiata salt-inducible-2) for functional characterization. Bioinformatics analysis revealed that SbSI-2 protein has predicted nuclear localization signals and a strong protein-protein interaction domain. Transient expression of the RFP:SbSI2 fusion protein confirmed that SbSI-2 is a nuclear-localized protein. Genomic organization study showed that SbSI-2 is intronless and has a single copy in Salicornia genome. Quantitative RT-PCR analysis revealed higher SbSI-2 expression under salt stress and desiccation conditions. The SbSI-2 gene was transformed in E. coli and tobacco for functional characterization. pET28a-SbSI-2 recombinant E. coli cells showed higher tolerance to desiccation and salinity compared to vector alone. Transgenic tobacco plants overexpressing SbSI-2 have improved salt- and osmotic tolerance, accompanied by better growth parameters, higher relative water content, elevated accumulation of compatible osmolytes, lower Na+ and ROS accumulation and lesser electrolyte leakage than the wild-type. Overexpression of the SbSI-2 also enhanced transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results demonstrate that SbSI-2 might play an important positive modulation role in abiotic stress tolerance. This identifies SbSI-2 as a novel determinant of salt/osmotic tolerance and suggests that it could be a potential bioresource for engineering abiotic stress tolerance in crop plants.

Figure 1. Genomic organization analysis.

(A) Genomic organization study, Lane 1: Amplified SbSI-2 PCR product from cDNA Lane 2: Amplified SbSI-2 PCR product from genomic DNA M: 100 bp DNA ladder (B) Southern blot of SbSI-2 gene from Salicornia brachiata genomic DNA. C: positive control (SbSI-2 gene cloned in pGEMT-T Easy vector).

Figure 2. Subcellular localization of RFP:SbSI-2 fusion protein in onion epidermal cells.

(A) Schematic representation of the pSITE-3CA-2X35S:RFP:SbSI-2 construct (RFP:SbSI-2) used for transient expression. (B) Cells with constructs expressing red fluorescence protein (RFP) alone and the RFP:SbSI-2 fusion protein were analyzed under bright and red fluorescence field. (C–D) Quantitative real-time PCR analysis of SbSI-2 under salt and desiccation conditions for different time period in S.brachiata. The relative fold expression of SbSI-2 at different time points under stress was calculated using the Ct value of untreated plants (control plant) at respective time points.

Figure 3. Growth analysis of recombinant E. coli cells having SbSI-2.

(A–D) Spot assay of BL21 (DE3)/pET28a-SbSI-2 and BL21 (DE3)/pET28a on LB basal plates and LB supplemented with NaCl, KCl and Mannitol. Ten microliters from 10⁻³ to 10⁻⁵ dilutions were spotted on (A) LB basal plates, (B) LB supplemented with 500 mM NaCl, (C) 500 mM KCl, and (D) 600 mM Mannitol. (E–I) Growth analysis of novel gene SbSI-2 was carried out in LB liquid medium with different supplements. (E) LB medium, (F) 500 mM NaCl, (G) 500 KCl, (H) 10% PEG, and (I) 600 mM Mannitol. O.D₆₀₀ was recorded at 2 h interval up to 12 h and mean values are represented in graph.

Figure 4. Confirmation of transgenic tobacco plants.

(A) Schematic representation of the pCAMBIA2301-35S:SbSI-2 construct used to transform tobacco plants with the SbSI-2 gene, (B) GUS assay of seedlings, showing positive GUS expression in the transgenic lines, (C) Transcript levels of the SbSI-2 gene in transgenic lines and WT plants via semi-quantitative RT-PCR, (D) Southern analysis of transgenic lines, (E–F) Germination of seeds from transgenic lines (L11, L17 and L22) and WT plants in (E) 0 mM, and (F) 200 mM NaCl and (G) Graphs represent the percentage germination of transgenic lines (L11, L17 and L22) and WT plants in salt stress and normal condition. Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 5. Phenotypic comparison of the growth of WT and transgenic lines overexpressing the SbSI-2 gene under salt stress.

(A–B) Growth comparison of transgenic lines (L11, L17 and L22) and WT seedlings after 30 days in (A) 0 mM, and (B) 200 mM NaCl. (C–D) Growth of whole plants from transgenic lines (L11, L17 and L22) and WT plants in (C) 0 mM and (D) 200 mM NaCl in culture bottles.

Figure 6. Comparison of growth parameters of seedlings from transgenic lines (L11, L17 and L22) and WT in 0 mM, and 200 mM NaCl.

(A) shoot length, (B) root length, (C) leaf area, (D) fresh weight, (E) dry weight and (F) relative water content (RWC). Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 7. Comparison of various biochemical and physiological parameters of transgenic lines (L11, L17 and L22) and WT under salt stress.

Chlorophyll (A), Electrolyte leakage (B), and proline (C) contents of transgenic (L11, L17 and L22) and WT seedlings grown in 0 mM, and 200 mM NaCl. (D–E) In vivo localization of O₂ ⁻ and H₂O₂ in seedlings of 35S:SbSI-2 transgenic lines and WT under salt stress. (D) Localization of O₂ ⁻ by NBT staining, (E) Localization of H₂O₂ by DAB staining. Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 8. Ion content analysis.

Na⁺ (A), K⁺ (B) and Ca²⁺ (D) contents in seedlings of transgenic lines (L11, L17 and L22) and WT grown in 0 mM, and 200 mM NaCl. Individual K⁺/Na⁺ ratios are shown in (C). Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 9. Phenotypic comparison of the growth of WT and transgenic lines overexpressing the SbSI-2 gene under osmotic stress.

(A–B) Germination of seeds from transgenic lines (L11, L17 and L22) and WT plants in (A) 0 mM, and (B) 300 mM mannitol. (C) Graphs represent the percentage germination of transgenic lines (L11, L17 and L22) and WT plants in osmotic stress (300 mM mannitol) and normal condition. (D–E) Growth comparison of transgenic lines (L11, L17 and L22) and WT seedlings in (d) 0 mM, and (e) 300 mM mannitol. Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 10. Comparison of growth parameters of seedlings from transgenic lines and WT in 0(osmotic stress).

(A) shoot length, (B) root length, (C) leaf area, (D) fresh weight, (E) dry weight and (F) relative water content (RWC). Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 11. Comparison of various biochemical and physiological parameters of transgenic lines (L11, L17 and L22) and WT under osmotic stress.

Chlorophyll content (A), Electrolyte leakage (B), and proline contents (C) of transgenic lines (L11, L17 and L22) and WT plants grown in the presence of 0 mM, and 300 mM mannitol. (D,E) In vivo localization of O₂ ⁻ and H₂O₂ in seedlings of 35S:SbSI-2 transgenic lines and WT under osmotic stress. (D) Localization of O₂ ⁻ by NBT staining, (E) Localization of H₂O₂ by DAB staining. Mean values that were significantly different at p≤0.05 within treatment from each other are indicated by different letters (a, b and c).

Figure 12. Expression analysis of ROS-related genes (NtSOD, NtCAT, NtAPX) in WT and SbSI-2-overexpressing plants by qRT-PCR.

(A, C and E) Expression analysis under salt stress (200 mM NaCl) and (B, D and F) Expression analysis under osmotic stress (300 mM mannitol).

Figure 13. Expression analysis of stress-related transcription factors (NtDREB2 and AP2-domain containing TF) in WT and SbSI-2-overexpressing plants by qRT-PCR.

(A and C) Expression analysis under salt stress (200 mM NaCl), and (B and D) Expression analysis under osmotic stress (300 mM mannitol).