Overexpression of SbSI-1, A Nuclear Protein from Salicornia brachiata Confers Drought and Salt Stress Tolerance and Maintains Photosynthetic Efficiency in Transgenic Tobacco

Abstract

A novel Salicornia brachiata Salt Inducible (SbSI-1) gene was isolated and overexpressed in tobacco for in planta functional validation subjected to drought and salt stress. SbSI-1 is a nuclear protein. The transgenic tobacco overexpressing SbSI-1 gene exhibited better seed germination, growth performances, pigment contents, cell viability, starch accumulation, and tolerance index under drought and salt stress. Overexpression of SbSI-1 gene alleviated the build-up of reactive oxygen species (ROS) and curtailed the ROS-induced oxidative damages thus improved the physiological health of transgenic tobacco under stressed conditions. The higher activities of antioxidant enzymes, lower accumulation of ROS, higher membrane stability, relative water content, and polyphenol contents indicated the better survival of the transgenic tobacco than wild-type (WT) tobacco under stressed conditions. Transgenic tobacco had a higher net photosynthetic rate, PSII operating efficiency, and performance index under drought and salt stress. Higher accumulation of compatible solutes and K⁺/Na⁺ ratio in transgenic tobacco than WT showed the better osmotic and redox homeostasis under stressed conditions. The up-regulation of genes encoding antioxidant enzymes (NtSOD, NtAPX, and NtCAT) and transcription factors (NtDREB2 and NtAP2) in transgenic tobacco under stressed conditions showed the role of SbSI-1 in ROS alleviation and involvement of this gene in abiotic stress tolerance. Multivariate data analysis exhibited statistical distinction among growth responses, physiological health, osmotic adjustment, and photosynthetic responses of WT and transgenic tobacco under stressed conditions. The overexpression of SbSI-1 gene curtailed the ROS-induced oxidative damages and maintained the osmotic homeostasis under stress conditions thus improved physiological health and photosynthetic efficiencies of the transgenic tobacco overexpressing SbSI-1 gene.

Keywords: abiotic stress; and transgenic; drought; halophyte; oxidative damage; salt.

Figure 1

Subcellular localization of SbSI-1 protein, homolog determination and molecular analysis of transgenic tobacco. Transient expression of RFP alone and RFP:SbSI-1 translational fusion protein in onion epidermal cells (A), determination of homologs of SbSI-1 in S. brachiata by southern blot analysis (B), PCR confirmation of transgenic tobacco by uidA and SbSI-1 gene amplification (C), semi-quantitative RT-PCR transgene-expression in transgenic tobacco using actin as an internal control (D), and determination of transgene integration in tobacco by southern blot analysis (E). The lane 1–2 are samples digested with EcoR 1 and Xho 1 respectively, PC is positive (SbSI-1 PCR product of ORF region) control, M is DNA ladder, WT-wild type plant/negative control and L_X are transgenic tobacco lines.

Figure 2

Seed germination in WT and transgenic lines under mannitol and NaCl stress after 21 days (A), total chlorophyll contents (B), and cell viability assay (C) of WT and transgenic tobacco under control and stress (PEG and NaCl) conditions for 7 days. The ^*, ^**, and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.05, 0.01, and 0.001 respectively.

Figure 3

Growth responses of WT and transgenic (L₈, L₂₂, and L₃₃) tobacco under drought (200 mM mannitol) and salt (200 mM NaCl) stress conditions for 21 days.

Figure 4

Leaf disc assay of WT and transgenic tobacco under control (A) and salt (B–100 mM, C–150 mM, D–200 mM NaCl) and drought (E–10% PEG) conditions.

Figure 5

Accumulation of starch in leaves (A), tolerance index (B), and in vivo detection of ${\text{O}}_2^{-}$ (C) and H₂O₂ (D) in leaves of WT and transgenic tobacco under control and stress (drought and salt) conditions. The ^* and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.05 and 0.001 respectively.

Figure 6

Comparison of activity SOD (A), APX (B), and CAT (C) in WT and transgenic tobacco under control and stress (drought and salt) conditions. The ^*, ^**, and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.05, 0.01, and 0.001 respectively.

Figure 7

Comparison of (A) lipid peroxidation, (B) electrolyte leakage, (C) membrane stability index, and (D) polyphenol contents in WT and transgenic tobacco under control and stress (drought and salt) conditions. The ^*, ^**, and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.05, 0.01, and 0.001 respectively.

Figure 8

Photosynthetic performances of WT and transgenic tobacco under control and stress (drought and salt) conditions. Comparison of net photosynthesis rate (A), stomatal conductance (B), intercellular CO₂ concentration (C), transpiration rate (D), PSII operating efficiency (E), electron transport rate (F), photochemical quenching (G), and non-photochemical quenching (H) in WT and transgenic tobacco under control and stress (drought and salt) conditions. The ^*, ^**, and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.05, 0.01, and 0.001 respectively.

Figure 9

Photosynthetic efficiency of WT and transgenic tobacco. Spider plots of photosynthetic fluxes depicting behavior of Photosystem II in leaves of WT and transgenic (L₈, L₂₂, and L₃₃) tobacco under control and stress (drought and salt) conditions.

Figure 10

Comparison of proline (A), total soluble sugar (B), reducing sugar (C), free amino acid (D) contents, and osmotic potential (E) in WT and transgenic tobacco under control and stress (drought and salt) conditions. The ^*, ^**, and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.05, 0.01, and 0.001 respectively.

Figure 11

Ionic contents. Comparison of sodium-potassium ratio in WT and transgenic tobacco under control and stress (drought and salt) conditions. The ^** and ^*** denote statistical significance in responses of transgenic lines against control at P value ≤0.01 and 0.001 respectively.