Overexpression of an alfalfa glutathione S-transferase gene improved the saline-alkali tolerance of transgenic tobacco

Abstract

Abiotic stresses restrict the productivity and quality of agricultural crops. Glutathione S-transferase (GST) utilizes glutathione to scavenge reactive oxygen species (ROS) that result from abiotic stresses. This study aimed to determine the expression pattern of the MsGSTU8 gene and its effects on saline-alkali tolerance. MsGSTU8, from alfalfa (Medicago sativa 'Zhaodong'), was transformed into transgenic tobacco (Nicotiana tabacum) and overexpressed to determine its effects on saline-alkali tolerance. The gene products in alfalfa localized to the cytoplasm and the transcript levels were higher in the leaves than the roots and stems. Expression was strongly induced by cold, drought, salt and saline-alkali stresses as well as abscisic acid (ABA) treatments. The transgenic tobacco lines had significantly higher transcription levels of the abiotic stress-related genes and higher GST activity than the wild types. Transgenic tobacco lines with saline-alkali treatments maintained their chlorophyll content, showed improved antioxidant enzyme activity and soluble sugar levels, reduced ion leakage, O₂ ^.-, H₂O₂ accumulation and malondialdehyde content. Our results indicate that overexpression of MsGSTU8 could improve resistance to saline-alkali stresses by decreasing the accumulation of ROS and increasing the levels of antioxidant enzymes. Furthermore, they suggest that MsGSTU8 could be utilized for transgenic crop plant breeding.

Keywords: Alfalfa; Antioxidant enzyme; MsGSTU8; ROS; Saline-alkali tolerance; Transgenic tobacco.

Fig. 1.

Phylogenetic analysis of MsGSTU8 with its homologous proteins from other plant species. The phylogenetic tree was constructed by Clustal X2 and MEGA 6.0 and performed with the Neighbor-Joining algorithm.

Fig. 2.

Subcellular localization of the MsGSTU8 protein. Transient expression of fusion protein MsGSTU8-GFP (p16318-MsGSTU8-GFP) and GFP (p16318-GFP) in Arabidopsis mesophyll protoplasts was analyzed by confocal laser scanning microscope. GFP, dark field; Bright, under light; Auto, red fluorescence indicates chloroplast autofluorescence; Merge, together with corresponding merged images. Scale bars: 5 µm.

Fig. 3.

Gene expression profile of MsGSTU8 in alfalfa. (A) The spatial-specific expression of MsGSTU8 in vegetative tissues of alfalfa. (B–F) Time-course expression patterns of MsGSTU8 in leaves and roots from alfalfa under cold treatment (B; 4°C), drought treatment (C; 400 mM mannitol), salt treatment (D; 300 mM NaCl), saline-alkali treatment (E; 100 mM Na₂CO₃: NaHCO₃, 1: 2), and treatment with 100 μM ABA (F) examined by qPCR. The relative abundance of the transcripts was determined by qPCR from total RNA of the indicated organs. Values represent mean±s.d. (n=3). Different letters represent significant differences at (P<0.05) according to LSD and Duncan's multiple range tests, and asterisks indicate significant difference from control (*P<0.01, **P<0.01, ***P<0.001).

Fig. 4.

The transcript and translation levels of MsGSTU8 in WT and transgenic tobacco. From left to right, the four-color histogram shows WT, L26, L27 and L37. (A) The transcript level of MsGSTU8 in MsGSTU8-overexpressing tobacco assayed by qPCR. For the assay, 4-week-old tobacco leaves were harvested as samples. The mRNA level of MsGSTU8 in WT plants were normalized as 1.0. (B) Enzymatic assay for GST using 4-week-old WT and transgenic tobacco lines under 0 mM or 30 mM NaHCO₃. FW, fresh weight. All experiments were repeated three times. Data are the mean±s.d. of three independent experiments. Different uppercase letters and lowercase letters represent significant differences at (P<0.05) according to LSD and Duncan's multiple range tests.

Fig. 5.

Tolerance of transgenic tobacco to saline-alkali stress. From left to right, the four-color histogram shows WT, L26, L27 and L37. (A) The phenotype of 4-week-old WT and transgenic tobacco line leaf discs under 30 mM NaHCO₃ treatment for 5 days. (B) Effect of normal condition and salt-alkaline stress on chlorophyll concentration in the leaf discs of WT and transgenic tobacco lines. The data presented are the means±s.d. of three biological replicates. Different uppercase letters and lowercase letters represent significant differences at (P<0.05) according to LSD and Duncan’s multiple range tests.

Fig. 6.

ROS accumulation in WT and transgenic tobaccos under saline-alkali stress. From left to right, the four-color histogram shows WT, L26, L27 and L37. Effects of normal condition and saline-alkali stress on O₂^.− productivity rate (A) and H₂O₂ concentrations (B) in the leaves of WT and transgenic tobacco lines. Each data column represents the mean (with s.d. bar) of three replicates. One-way ANOVA post hoc multiple comparisons, LSD and Duncan's multiple range tests were selected simultaneously in equal variances assumed. The different uppercase letters and lowercase letters represent significant differences (P<0.05) according to LSD and Duncan's multiple range tests. The different uppercase letters represent significant differences in sample data of both control and treatment group (P<0.05). Different lowercase letters represent significant differences of the sample data of control or treatment group (P<0.05).

Fig. 7.

MsGSTU8 transgenic tobacco plants show enhanced antioxidant enzyme activity under saline-alkali treatment. From left to right, the four-color histograms show WT, L26, L27 and L37. (A–C) Variations in activity of SOD (A), POD (B) and CAT (C) in the leaves of WT and transgenic tobacco lines treated with 30 mM NaHCO₃ in Hoagland's solution. Bars indicate standard deviations of three replicates. One-way ANOVA post hoc multiple comparisons, LSD and Duncan's multiple range tests were selected simultaneously in equal variances assumed. The different uppercase letters and lowercase letters represent significant differences (P<0.05) according to LSD and Duncan's multiple range tests. The different uppercase letters represent significant differences in sample data of both control and treatment group (P<0.05). Different lowercase letters represent significant differences of the sample data of control or treatment group (P<0.05).

Fig. 8.

MsGSTU8 conferred enhanced saline-alkali stress resistance by protecting the membrane from damage and maintaining osmotic pressure in transgenic tobacco. From left to right, the four-color histograms show WT, L26, L27 and L37. (A) Effect of normal condition and saline-alkali stress on ion leakage in the leaves of WT and transgenic tobacco lines. Comparison of physiological indices between the WT and transgenic tobacco lines under saline-alkali stress. After saline-alkali treatments, the leaves of 4-week-old WT and transgenic tobacco lines were collected to measure the (B) MDA and (C) soluble sugar contents. Data are shown as the means±s.d. calculated from three biological replicates. Different uppercase letters and lowercase letters represent significant differences (P<0.05) according to LSD and Duncan’s multiple range tests.

Fig. 9.

Comparison of stress-related gene expression levels between the WT and transgenic tobacco lines after saline-alkali treatment. The y-axis records the relative gene expression levels with NtGAPDH as the endogenous reference. Data are shown as the means±s.d. calculated from three biological replicates. Different letters represent significant differences at (P<0.05) according to LSD and Duncan's multiple range tests.