Overexpression of a S-Adenosylmethionine Decarboxylase from Sugar Beet M14 Increased Araidopsis Salt Tolerance

Abstract

Polyamines play an important role in plant growth and development, and response to abiotic stresses. Previously, differentially expressed proteins in sugar beet M14 (BvM14) under salt stress were identified by iTRAQ-based quantitative proteomics. One of the proteins was an S-adenosylmethionine decarboxylase (SAMDC), a key rate-limiting enzyme involved in the biosynthesis of polyamines. In this study, the BvM14-SAMDC gene was cloned from the sugar beet M14. The full-length BvM14-SAMDC was 1960 bp, and its ORF contained 1119 bp encoding the SAMDC of 372 amino acids. In addition, we expressed the coding sequence of BvM14-SAMDC in Escherichia coli and purified the ~40 kD BvM14-SAMDC with high enzymatic activity. Quantitative real-time PCR analysis revealed that the BvM14-SAMDC was up-regulated in the BvM14 roots and leaves under salt stress. To investigate the functions of the BvM14-SAMDC, it was constitutively expressed in Arabidopsis thaliana. The transgenic plants exhibited greater salt stress tolerance, as evidenced by longer root length and higher fresh weight and chlorophyll content than wild type (WT) under salt treatment. The levels of spermidine (Spd) and spermin (Spm) concentrations were increased in the transgenic plants as compared with the WT. Furthermore, the overexpression plants showed higher activities of antioxidant enzymes and decreased cell membrane damage. Compared with WT, they also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. Together, these results suggest that the BvM14-SAMDC mediated biosynthesis of Spm and Spd contributes to plant salt stress tolerance through enhancing antioxidant enzymes and decreasing ROS generation.

Keywords: ROS; S-adenosylmethionine decarboxylase; antioxidant enzyme; salt stress; sugar beet.

Figure 1

Ecoli expression of the recombinant sugar beet M14 S-adenosylmethionine decarboxylase (BvM14-SAMDC) protein. (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of bacterial expression of BvM14-SAMDC. Total soluble protein fractions with the empty vector after 4 h isopropyl β-d-thiogalactoside (IPTG) induction (lanes 1 and 2); and total soluble protein fractions with pET28a-BvM14-SAMDC after 4 h IPTG induction (lanes 3 and 4). (b) Immunoblot identification of the purified recombinant BvM14-SAMDC using anti-His antibody. Arrows indicate the BvM14-SAMDC position.

Figure 2

BvM14-SAMDC expression patterns in different organs and in response to salt stress treatments. (a) BvM14-SAMDC expression profiles in different organs. (b) leaves and (c) roots of the M14 plants treated with 400 mM NaCl for different time periods. Data are the means of three biological replicates with standard deviation (SD) bars.

Figure 3

Effect of salt stress on seedling growth phenotype, root length, fresh weight, chlorophyll and electrical conductivity in wild type (WT), BvM14-SAMDC-overexpressed in wild type Arabidopsis (OX), atsamdc1 mutant (KD) and transgenic BvM14-SAMDC in the mutant seedlings (CO) leaves. (a) 8-day-old WT and transgenic seedlings, grown on MS medium, were transferred to new MS solid agar plates supplemented with 0 or 100 mM NaCl, followed by growth for 10 days; (b) root length; (c) fresh weight; (d) chlorophyll level; and (e) electrical conductivity in control and 100 mM NaCl treated seedlings. Different letters indicate significantly different at P < 0.05. Three biological replicates were performed.

Figure 4

Effects of salt stress on antioxidant enzyme system in the leaves of wild type (WT), BvM14-SAMDC-overexpression in WT Arabidopsis (OX), atsamdc1 mutant (KD) and transgenic BvM14-SAMDC in the mutant seedlings (CO). (a) leaf malondialdehyde (MDA) content; (b–d) antioxidant enzyme activities under control and salt stress (100 mM NaCl) conditions. One unit of CAT activity was defined as the amount of enzyme required for l μmol of H₂O₂ decomposed within 1 min. One unit of SOD activity was defined as the amount of enzyme required for inhibition of photochemical reduction of r-nitro blue tetrazolium chloride (NBT) by 50%. One unit of POD activity was defined as the amount of enzyme required for oxidation of 5 μmol of guaiacol within 1 min. Different letters indicate significantly different at P < 0.05. Three biological replicates were performed.

Figure 5

Effects of salt stress on polyamine metabolism in the leaves of wild type (WT), BvM14-SAMDC-overexpression in WT Arabidopsis (OX), atsamdc1 mutant (KD) and transgenic BvM14-SAMDC in the mutant seedlings (CO). (a) SAMDC activity; (b) levels of putrescine (Put); (c) levels of spermine (Spm); and (d) levels of spermidine (Spd) under control and salt stress (100 mM NaCl) conditions. One unit of SAMDC activity was defined as the amount of enzyme required for catalyzing 2 μmol SAM within 1 min. Different letters indicate significantly different at P < 0.05. Three biological replicates were performed.

Figure 6

Effects of salt stress on polyamine oxidase (PAO) activity, H₂O₂ content and mRNA levels of RbohD and RbohF in the leaves of wild type (WT) and BvM14-SAMDC-overexpression in WT Arabidopsis (OX). (a) PAO activity; (b) H₂O₂ content; (c) mRNA levels of RbohD; and (d) mRNA levels of RbohF under control and salt stress (100 mM NaCl) conditions. One unit of PAO activity was defined as the amount of enzyme required for catalyzing 1 mmol of Spd or Spm oxidation within 1 min. Different letters indicate significantly different at P < 0.05. Three biological replicates were performed.

Figure 7

Overview diagram showing how the BvM14-SAMDC functions in mediating plant salt stress tolerance. Salt stress can turn on the expression of BvM14-SAMDC, which plays an important role in the biosynthesis of PAs. PAs may inhibit the expression of ROS-biosynthetic enzymes and activate ROS-detoxifying enzymes, leading to reduced ROS levels and enhanced salt stress tolerance phenotype. SAMDC, S-adenosylmethionine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase; tSPMS, thermospermine synthase; dcSAM, decarboxylated S-adenosylmethionine; Put, putrescine; SAM, S-adenosylmethionine; Spd, spermidine; Spm, spermine.