A novel α/β-hydrolase gene IbMas enhances salt tolerance in transgenic sweetpotato

Abstract

Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to α/β-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity.

Figure 1. Sequence alignment of IbMas protein with its homologous proteins from various plant species.

The proteins are as follows: XP_006363736 from Solanum tuberosum, XP_007039974 from Theobroma cacao, XP_002267811 from Vitis vinifera, XP_004245715 from Solanum lycopersicum, EXC17874 from Morus notabilis, XP_006368699 from Populus trichocarpa, KDP25601 from Jatropha curcas and XP_002509605 from Ricinus communis. The maspardin domain is marked by asterisk line.

Figure 2. Phylogenetic tree of IbMas protein with its homologous proteins.

The branch lengths are proportional to distance.

Figure 3. Expression analysis of the IbMas gene in sweetpotato line ND98 by real-time quantitative PCR.

(A) and (B) Relative expression level of IbMas in ND98 after different times (h) of 200 mM NaCl and 100 µM ABA treatment, respectively. The 4-week-old in vitro-grown plants of ND98 were submerged in 1/2 MS medium containing 200 mM NaCl and 100 µM ABA, respectively, and sampled at 0, 3, 6, 12, 24 and 48 h after treatment to analyze the expression of IbMas. The sweetpotato β-actin gene was used as an internal control. Data are presented as means ± SE (n = 3).

Figure 4. Production of transgenic sweetpotato plants overexpressing the IbMas gene.

(A) Embryogenic suspension cultures rapidly proliferating in MS medium containing 2.0 mg L⁻¹ 2,4-D. (B) PPT-resistant calluses formed on MS medium with 2.0 mg L⁻¹ 2,4-D, 100 mg L⁻¹ Carb and 0.8 mg L⁻¹ PPT after 8 weeks of selection. (C) Regeneration of plantlets from PPT-resistant calluses on MS medium with 1.0 mg L⁻¹ ABA, 100 mg L⁻¹ Carb and 0.8 mg L⁻¹ PPT. (D) PCR analysis of transgenic plants. Lane M: DL2000 DNA marker; Lane W: water as negative control; Lane P: plasmid pCAMBIA3301-IbMas as positive control; Lane WT: wild-type as negative control; Lanes L39, L52, L55, L99, L101, L102 and L109: transgenic plants. (E) and (F) WT and transgenic plants grown in a greenhouse, respectively. (G) and (H) WT and transgenic plants grown in a field, respectively. (I) and (J) Storage roots of WT and transgenic plants, respectively.

Figure 5. Responses of the IbMas-overexpressing sweetpotato plants under 86 mM NaCl stress.

(A) The growth and rooting of trangenic plants and wild-type plant (WT) cultured for 4 weeks on MS medium supplemented with 86 mM NaCl. (B) and (C) Phenotypes of salt-tolerant transgenic plants (L52, L99, L102 and L101) and WT incubated for 4 weeks in Hoagland solution with 86 mM NaCl; all of the cuttings of L52, L99, L102 and L101 formed obvious new leaves and roots and those of WT died.

Figure 6. Expression analysis of IbMas gene in the transgenic sweetpotato plants by real-time quantitative PCR.

The 36 transgenic and wild-type (WT) in vitro-grown plants were submerged in 1/2 MS medium with 200 mM NaCl for 12 h to analyze the expression of IbMas. The sweetpotato β-actin gene was used as an internal control. The results are expressed as relative values based on WT as reference sample set to 1.0. Data are presented as means ± SE (n = 3). * and ** indicate a significant difference from that of WT at P<0.05 and <0.01, respectively, by Student's t-test.

Figure 7. Phenotypes of the IbMas-overexpressing sweetpotato plants grown in pots under 200 mM NaCl stress.

The 25-cm-long cuttings of the salt-tolerant transgenic plants (L52, L99, L102 and L101) and wild-type plants (WT) were grown in 19-cm diameter pots containing a mixture of soil, vermiculite and humus (1∶1∶1, v/v/v) in a greenhouse, with one cutting per pot. All pots were irrigated sufficiently with half-Hoagland solution for 10 days until the cuttings formed new leaves, and then each pot was irrigated a 200 mL of 0 and 200 mM NaCl solution, respectively, once every 2 days for 4 weeks. All of L52, L99, L102 and L101 plants showed good growth and increased physical size and those of WT died under 200 mM NaCl stress.

Figure 8. Biomass of the IbMas-overexpressing sweetpotato plants grown in pots under 200 mM NaCl stress.

The 25-cm-long cuttings of the salt-tolerant transgenic plants (L52, L99, L102 and L101) and WT were grown in 19-cm diameter pots containing a mixture of soil, vermiculite and humus (1∶1∶1, v/v/v) in a greenhouse, with one cutting per pot, and treated as described in Fig. 6. After treatment, the plant fresh weight (FW) was measured immediately. The plants were then dried for 24 h in an oven at 80°C and weighed (DW). All treatments were performed in triplicate. Data are presented as means ± SE (n = 3). * and ** indicate a significant difference from that of WT at P<0.05 and <0.01, respectively, by Student's t-test.

Figure 9. Photosynthetic performance of the IbMas-overexpressing sweetpotato plants under salt stress.

(A), (B), (C) and (D) Photosynthetic rate, stomatal conductance, transpiration rate and chlorophyll relative content, respectively, in the leaves of salt-tolerant transgenic plants (L52, L99, L102 and L101) and wild-type plant (WT). The 25-cm-long cuttings of the salt-tolerant transgenic plants evaluated with water culture assay and WT were grown in 19-cm diameter pots containing a mixture of soil, vermiculite and humus (1∶1∶1, v/v/v) in a greenhouse, with one cutting per pot. All pots were irrigated sufficiently with half-Hoagland solution for 10 days until the cuttings formed new leaves, and then each pot was irrigated with a 200 mL of 200 mM NaCl solution once every 2 days for 2 weeks. Data are presented as means ± SE (n = 3). * and ** indicate a significant difference from that of WT at P<0.05 and <0.01, respectively, by Student's t-test.

Figure 10. Effects of salt stress on H₂O₂ accumulation in the IbMas-overexpressing sweetpotato plants.

(A) Accumulation of H₂O₂ in the leaves of salt-tolerant transgenic plants (L52, L99, L102 and L101) and wild-type plant (WT). A plant grown in a 19-cm diameter pot was irrigated with a 200 mL of 200 mM NaCl solution once every 2 days for 2 weeks. (B) The average intensity of DAB staining leaves after converting to 256 grey scale images. Data are presented as means ± SE (n = 3). * and ** indicate a significant difference from that of WT at P<0.05 and <0.01, respectively, by Student's t-test.

Figure 11. Relative expression level of IbMas and salt stress responsive genes in the IbMas-overexpressing sweetpotato plants.

P5CS: pyrroline-5-carboxylate synthase; P5CR: pyrroline-5-carboxylate reductase; SOD: superoxide dismutase; psbA: encoding D1 protein; PRK: phosphoribulokinase (PRKase). The salt-tolerant transgenic plants (L52, L99, L102 and L101) and wild-type plant (WT) in vitro-grown plants were submerged for 12 h in 1/2 MS medium containing 0 and 200 mM NaCl, respectively. The sweetpotato β-actin gene was used as an internal control. The results are expressed as relative values based on WT grown under control condition as reference sample set to 1.0. Data are presented as means ± SE (n = 3). * and ** indicate a significant difference from that of WT at P<0.05 and <0.01, respectively, by Student's t-test.