Overexpression of S-Adenosyl-l-Methionine Synthetase 2 from Sugar Beet M14 Increased Arabidopsis Tolerance to Salt and Oxidative Stress

Abstract

The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH). A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS), is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM), a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H₂O₂ tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H₂O₂ stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H₂O₂ tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet.

Keywords: ">l-methionine synthetase; S-adenosyl-; antioxidant system; polyamine; salt stress; sugar beet M14.

Figure 1

Experimental scheme of screening differentially expressed genes by suppression subtractive hybridization (SSH) and functional classification of the identified genes using the UniProt database. (a) Experimental scheme; (b) Functional classification of the differential genes; EST stands for expressed sequence tag.

Figure 2

Tissue specific expression of BvM14-SAMS2 gene in the M14 plants and induction of the BvM14-SAMS2 mRNA in response to salt stress. (a) Real time-PCR detection of BvM14-SAMS2 gene in different tissues. Time-course analysis of BvM14-SAMS2 relative expression levels in leaves (b); and roots (c) of the M14 plants under 200 mM NaCl stress. Data for real time-PCR analysis are the means of three biological replicates and three technology replicates (standard deviation, SD), separately. Each replicate contains five sugar beet seedlings. The 18S rRNA gene was used as the internal control for relative expression analysis.

Figure 3

Identification of atsams3 mutant and overexpressed BvM14-SAMS2 in Arabidopsis plants. (a) quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression levels of overexpressed BvM14-SAMS2 (OX1 and OX2) in Arabidopsis plants; (b) Structure of the AtSAMS3 gene. The T-DNA insertional site was 756 bp upstream of the start codon, as indicated by a triangle. The primers used to identify the T-DNA insertion were marked with arrows; (c) PCR analysis of the T-DNA insertion in the atsams3 mutant (KO); (d) RT-PCR analysis of the expression levels of AtSAMS3 in atsams3 mutant and wild type (WT); (e) Real-time PCR analysis of the expression levels of AtSAMS3 in the atsams3 mutant; (f) The concentration of S-adenosylmethionine (SAM) in 20-day old whole seedlings of wild type (WT), overexpressed BvM14-SAMS2 (OX1 and OX2), AtSAMS3 konckdown mutant (KO) and BvM14-SAMS2 in mutant complementation seedlings (CO). Data are the means of three biological replicates (SD) and each replicate contains five seedlings. Different letters indicate significant difference at p < 0.05.

Figure 4

Analysis of salt and H₂O₂ tolerance in transgenic Arabidopsis plants in comparison with the wild type and atsams3 mutant. (a) Phenotypes of wild type (WT), BvM14-SAMS2 BvM14-SAMS2-overexpressed seedlings in wild type Arabidopsis (OX), atsams3 mutant (KO), and BvM14-SAMS2 in mutant complementation seedlings (CO) under control and stress conditions. Photographs were taken 14 days after treatment. Inhibition of root length (b); and loss of fresh weight (c) were determined. Data are the means of three biological replicates (SD) and each replicate contains five seedlings. Different letters indicate significant difference at p < 0.05.

Figure 5

Analysis of salt and H₂O₂ tolerance in transgenic Arabidopsis plants in comparison with the wild type and atsams3 mutant in soil. (a) Phenotypes of wild type (WT), BvM14-SAMS2 BvM14-SAMS2-overexpressed seedlings in wild type Arabidopsis (OX), atsams3 mutant (KO), and BvM14-SAMS2 in mutant complementation seedlings (CO) under conditions of control and stress in soil. Photographs were taken 7 days after treatment; Total chlorophyll levels (b) were determined. Data are the means of three biological replicates (SD) and each replicate contains five seedlings. Different letters indicate significant difference at p < 0.05.

Figure 6

Effects of salt and H₂O₂ stresses on antioxidant system activity H₂O₂ content (a); malondialdehyde (MDA) content (b); and antioxidant enzyme activities (c–e) were measured in wild type (WT), transgenic BvM14-SAMS2 wild type (WT), BvM14-SAMS2-overexpressed in wild type Arabidopsis (OX), atsams3 mutant (KO) and transgenic BvM14-SAMS2 in the mutant seedlings (CO) leaves. Data are the means of three biological replicates (SD) and each replicate contains five seedlings. Different letters indicate significant difference at p < 0.05.

Figure 7

Effects of salt and H₂O₂ stresses on polyamines (PAs). Total putrescine (Put) content (a); total spermidine (Spd) content (b); total spermine (Spm) content (c) in wild type (WT), transgenic BvM14-SAMS2 seedlings in wild type Arabidopsis (OX), atsams3 mutant (KO) and BvM14-SAMS2 in mutant complementation seedlings (CO) leaves. Data are the means of three biological replicates with standard deviation (SD), and each replicate contains five seedlings. Different letters indicate significant difference at p < 0.05.