Expression of the ggpPS gene for glucosylglycerol biosynthesis from Azotobacter vinelandii improves the salt tolerance of Arabidopsis thaliana

Abstract

Many organisms accumulate compatible solutes in response to salt or desiccation stress. Moderate halotolerant cyanobacteria and some heterotrophic bacteria synthesize the compatible solute glucosylglycerol (GG) as their main protective compound. In order to analyse the potential of GG to improve salt tolerance of higher plants, the model plant Arabidopsis thaliana was transformed with the ggpPS gene from the gamma-proteobacterium Azotobacter vinelandii coding for a combined GG-phosphate synthase/phosphatase. The heterologous expression of the ggpPS gene led to the accumulation of high amounts of GG. Three independent Arabidopsis lines showing different GG contents were characterized in growth experiments. Plants containing a low (1-2 micromol g(-1) FM) GG content in leaves showed no altered growth performance under control conditions but an increased salt tolerance, whereas plants accumulating a moderate (2-8 micromol g(-1) FM) or a high GG content (around 17 micromol g(-1) FM) showed growth retardation and no improvement of salt resistance. These results indicate that the synthesis of the compatible solute GG has a beneficial effect on plant stress tolerance as long as it is accumulated to an extent that does not negatively interfere with plant metabolism.

Fig. 1.

Expression of the ggpPS gene from Azotobacter vinelandii in the transgenic lines AtAF-19, AtAF-30, and At-AF-4 obtained after transformation of Arabidopsis thaliana Col-0 wild type (WT) plants. (A) Schematic representation of the T-DNA region transferred into A. thaliana. (B) Electrophoretically separated DNA fragments obtained by PCR using specific primer combinations for nptII (lanes 1, Kan-A/-S), ggpPS (lanes 2, AvggpS-5′/-3′), and a T-DNA part at the left border (lanes 3, TDNA-AFfw/-rev) with DNA isolated from plants of the wild type (WT), transgenic lines AtAF-19, AtAF-30, and AtAF-4. (C) Expression of the ggpPS gene in plants of the wild type (WT) and transgenic plants of the lines AtAF-19, AtAF-30, and AtAF-4. The ggpPS mRNA was detected using a ³²[P]-labelled probe. The signal intensity of the 18S rRNA was used as loading control. Relative expression of the ggpPS gene in the transgenic lines was calculated by dividing the signal intensity for the ggpPS mRNA by the signal intensity of the 18S rRNA. Results from one representative experiment are shown. Abbreviations: BL, border left; BR, border right; P35S, cauliflower mosaic virus 35S promoter; dP35S, tandem cauliflower mosaic virus 35S promoter; T35S, cauliflower mosaic virus 35S terminator; nptII, neomycin phosphotransferase; ggpPS, combined glucosylglycerol-phosphate phosphatase/synthase from A. vinelandii; M, marker.

Fig. 2.

Accumulation of glucosylglycerol (GG) in plants of the wild type (WT) and the selected transgenic lines AtAF-19, AtAF-30, and AtAF-4 at different developmental stages and in different organs. Plants were grown under standard conditions on soil. Soluble sugars were extracted from plant material and analysed by gas chromatography. (A) Amount of GG in rosette leaves of the WT and the transgenic lines AtAF-19, AtAF-30, and AtAF-4. Plant material was collected before primary inflorescences appeared (growth stage 5.10). These data are means ±SD of five independent experiments each with three pots per line. (B) GG content in rosette leaves of the WT and the transgenic lines during plant development. (C) GG content in different plant organs and tissues of flowering plants (growth stage 6.50). Arabidopsis growth stages were defined according to Boyes et al. (2001). The data in (B) and (C) are means ±SD of triplicates from one representative experiment.

Fig. 3.

Phenotypic appearance of Arabidopsis wild-type plants (WT) and transgenic lines showing various levels of GG accumulation (AtAF-19, AtAF-30, AtAF-4). (A) Photograph of representative individuals taken after growth at standard conditions for 45 d (growth stage 6.50; Boyes et al., 2001). (B, C) Total leaf number and the maximum rosette radius (B) as well as total plant biomass (C) were estimated for 10 different individual plants of each line (* P <0.05, ** P <0.01).

Fig. 4.

Germination of seeds and survival of seedlings from Arabidopsis plants of the wild type (WT) and of the transgenic lines showing different levels of GG accumulation (AtAF-19, AtAF-30, AtAF-4) at increasing NaCl concentrations. (A) Rate of seed germination under salt stress conditions (amount of added NaCl is given inside each panel). (B) GG and sucrose accumulation in seeds. (C) Survival rate of seedlings under standard conditions (left) and in the presence of 200 mM NaCl (right; * P <0.05). Data are the means ±SD of five independent experiments. In each experiment 36 seeds or seedlings per line were incubated on the same agar plate.

Fig. 5.

Comparison of growth of Arabidopsis plants of the wild type (WT) and the transgenic, GG-accumulating lines (AtAF-19, AtAF-30, AtAF-4) in the presence of different NaCl concentrations. (A) The increase in rosette leaf biomass was estimated by analysing plants before (growth stage 5.10) and 13 d after treatment with various NaCl solutions. In these experiments, WT and transgenic plants were grown in one pot to ensure similar growth and stress conditions. These data are means ±SD of two independent experiments each with five pots per line. (B) Photographs of representative plants were taken at the 13th day after salt-stress treatment (left WT plant, right transgenic plant). (C, D) Comparison of root growth by cultivating seedlings on MS agar medium containing various NaCl amounts in a vertically orientated position for at least 2 weeks. The root growth was calculated as relative values of primary root length of transgenic plants divided by mean root length of WT plants on the same plate (C). The quantitative data are means ±SD of two independent experiments each with 10 seedlings per line. The photograph shows seedlings and roots incubated for 2 weeks (D).