Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth

Abstract

Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0) and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis.

Figure 1. Expression analyses of FAD2 in wild-type Col-0 plants.

(A) and (B) RT-PCR analyses of FAD2 transcripts. R, root; RL, rosette leaf; CL, cauline leaf; St, stem; F, flower; Si, silique. (C) GUS expression in ProFAD2:GUS transgenic plants. The upper panels show a 1-, 2-, 3-, 7-, 14-day-old seedling, and a rosette leaf (T) of five-week-old plants, respectively. The lower panels depict root (R), rosette leaf (RL), cauline leaf (CL), stem (St), flower (F) and silique (Si) of five-week-old plants, respectively. Red arrows indicate the high GUS expression in trichomes. Scale bar  =  1 mm. (D) Quantitative real-time PCR analyses of FAD2 upon treatment with different stresses. Eight-day-old seedlings were treated with 300 mM NaCl or 300 mM mannitol. The unstressed expression level was assigned a value of 1. Data represents the average of three independent experiments ±SE.

Figure 2. Heterologous expression of Arbidopsis FAD2 in S. cerevisiae.

Cells of the wild-type yeast strain W303-1 were transformed with pVTFAD2Δ (FAD2Δ), pVTFAD2 (FAD2), pVTHaFAD2-1 (HaFAD2-1) and pVTHaFAD2-3 (HaFAD2-3), respectively. The mutated FAD2Δ gene was used as negative controls, whereas the sunflower HaFAD2-1 and HaFAD2-3 were used as positive controls. Photos were taken at 2 (YPD and 0.5 M) or 5 (1 M and 1.5 M) days. Photos are representatives of three independent experiments. WT, wild-type W303-1; FAD2Δ, W303-1 harboring the mutated FAD2Δ gene; FAD2, W303-1 harboring the functional FAD2 gene; HaFAD2-1 and HaFAD2-3, W303-1 harboring the functional sun flower FAD2 gene.

Figure 3. FAD2 gene mutation and fatty acid analyses in fad2.

(A) Intron-exon organization of the Arabidopsis FAD2 gene (not to scale). The mutation site was indicated. Solid boxes and lines represent exons and introns, respectively. (B) The amino acid mutation site in the predicted topology of FAD2 protein. Three His boxes (black ellipses) oriented on the cytosolic side of the membrane coordinate two iron atoms (gray circles) at the active site of the protein. The mutation site was indicated (from A₁₀₄ to T₁₀₄). (C) Fatty acid analyses of Col-0, fad2 and fad2 mutant complemented with the functional wild-type FAD2 or the mutated FAD2Δ gene. Col-0, wild-type Columbia; fad2, FAD2 mutant; fad2/FAD2-1 and fad2/FAD2-2, two transgenic fad2 lines complemented with the functional Col-0 FAD2 gene; fad2/FAD2Δ-1 and fad2/FAD2Δ-2, two transgenic fad2 lines complemented with the mutated FAD2Δ gene. Results are presented as means and standard errors from three independent experiments. ** indicates significant difference in comparison to Col-0 at P<0.01 (Student's t-test).

Figure 4. Germination of Col-0, fad2 and fad2/FAD2 under salt treatments.

(A) Seeds were sowed on MS medium supplemented with 0 (left), 100 (middle) or 125 mM NaCl (right). Photos were taken seven days after stratification. (B)–(E) Percentage of germinating seeds grown on MS medium supplemented with 0 (B), 75 (C), 100 (D) or 125 (E) mM NaCl. F, Percentages of germinating seeds grown on MS medium supplemented with different concentrations of NaCl three days after stratification. Results are presented as means and standard errors from three independent experiments (≥100 seeds of each line were sown for each experiment).

Figure 5. Stress response of Col-0, fad2 and fad2/FAD2.

Col-0, wild-type Columbia; fad2, FAD2 mutant; fad2/FAD, transgenic fad2 line 1 complemented with the Col-0 FAD2 gene (fad2/FAD2-1). (A) Phenotypes on MS medium supplemented with different concentrations of NaCl or mannitol. Photos were taken 7 days after the initiation of the treatments. B and C, Root growth on MS medium supplemented with different concentrations of NaCl (B) or mannitol (C) from (A). The relative root lengths were measured on day 7 after stratification (n = 60 for each condition). (D) Root elongations on MS medium supplemented with different concentrations of NaCl. Four-day-old seedlings were transferred to MS medium supplemented with 0, 75, 100 or 125 mM NaCl. Results are presented as means and standard errors from three independent experiments. * and ** indicate significant differences in comparison to Col-0 at P<0.05 and P<0.01, respectively (Student's t-test).

Figure 6. Survival rrates of Col-0, fad2 and fad2/FAD2.

(A)-(C), Five-day-old seedlings of Col-0, fad2 and fad2/FAD2 (fad2/FAD2-1) were transferred to MS medium supplemented with 0 (A), 200 (B), or 250 mM NaCl (C). Photos were taken 10 days after the initiation of the stress treatments. (n = 240). (D) Survival rates of plants in (B) and (C). Results are presented as means and standard errors from three independent experiments. ** indicates significant difference in comparison to wild-type at P<0.01 (Student's t-test) (n = 240).

Figure 7. Na⁺/K⁺ Ratio analyses in Col-0 and fad2 mutant.

Results are presented as means and standard errors from three independent experiments. ** indicates significant difference in comparison to Col-0 at P<0.01 (Student's t-test). Eight-day-old seedlings grown on MS medium supplemented with or without 75 mM NaCl were used.

Figure 8. Fatty acid composition and Na⁺/H⁺ exchanger activity assays.

(A) Fatty acid compositions. (B) Na⁺-dependent H⁺ exchange. TM control, tonoplast vesicles without NaCl treatment (upper); TM NaCl, tonoplast vesicles with NaCl treatment (middle); PM NaCl, plasma membrane vesicles with NaCl treatment (lower). (C) and (E) Rates of H⁺ translocation of tonoplast (C) and plasma membrane (E) vesicles. (D) and (F) Rates of Na⁺-dependent H⁺ effluxes in tonoplast (D) and plasma membrane (F) vesicles. Tonoplast and plasma membrane vesicles were isolated from the leaves of 4-week-old seedlings treated with or without 250 mM NaCl for 3 days. Col-0, wild-type Columbia; fad2, FAD2 mutant; fad2/FAD, fad2/FAD2-1. Results are presented as means and standard errors from four independent experiments. ** and ** indicate significant difference in comparison to wild-type at P<0.01 and P<0.001 (Student's t-test).

Figure 9. Intracellular Na⁺ distribution and Na⁺ extrusion in roots of Col-0, fad2 and fad2/FAD2.

(A)-(C) Representative examples (n = 10–12) of CoroNa-Green staining of the root tip region before and after NaCl treatment. (D) Magnifications of marked regions (red boxes) in (A) to (C). Red circles and white arrows indicate the vacuolar and cytoplamic regions, respectively. (E) and (F) Fluorescent intensities in the vacuole and cytosol were calculated for comparison. Error bars indicate SD of at least 100 cells randomly measured from 10 individual seedlings. (G) Net Na⁺ efflux in root tips. Inserted section show the mean flux rates within the measuring period of 18–20 min in NaCl-treated seedlings. ** indicates significant difference in comparison to wild-type at p<0.01 (Student's t-test). Roots of five-day-old seedlings were stained with CoroNa-Green AM after 72 h of 100 mM NaCl treatment and observed with a confocal microscope.