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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (7): 1017-1028.doi: 10.3724/SP.J.1006.2019.84142

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Ectopic expression of S-adenosylmethionine decarboxylase (GhSAMDC1) in cotton enhances salt tolerance in Arabidopsis thaliana

TIAN Wen-Gang1,ZHU Xue-Feng1,SONG Wen1,CHENG Wen-Han2,XUE Fei1,ZHU Hua-Guo1,*()   

  1. 1 College of Agronomy, Shihezi University / Key Oasis Eco-Agriculture Laboratory of Production and Group, Shihezi 832003, Xinjiang, China
    2 Jingchu University of Technology, Jingmen 448000, Hubei, China
  • Received:2018-11-05 Accepted:2019-01-19 Online:2019-07-12 Published:2019-03-15
  • Contact: Hua-Guo ZHU E-mail:57530422@qq.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31301363);This study was supported by the National Natural Science Foundation of China(31660427);Science and Technology Development Program of Xinjiang Production and Construction Groups Project(2015AC007);the Natural Science Foundation of Hubei Province(2017CFB162)

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Abstract:

Transgenic Arabidopsis thaliana (GhSAMDC1) was used to study the effect of overexpression of GhSAMDC1 on salt tolerance of Arabidopsis thaliana seedlings, Contents of endogenous polyamines, hydrogen peroxide (H2O2), malondialdehyde (MDA), and chlorophyll, ion permeability, antioxidant enzymes (SOD, CAT, POD) activities and expression levels were investigated under salt stress. The overexpression of GhSAMDC1 decreased the content of endogenous putrescine (Put) and increased spermidine (Spd) and spermine (Spm) contents in Arabidopsis thaliana. Under salt stress, the expression levels of spermidine synthase (AtSPDS1, AtSPDS2) and spermine synthase (AtSPMS) in transgenic lines were significantly higher than those in wild type, the contents of Spd and Spm were further increased, and the contents of H2O2, MDA, chlorophyll, and ion permeability were obviously decreased. Compared with the wild type, Transgenic lines had no remarkable difference in peroxidase (POD) activity, but significantly higher superoxide dismutase (SOD) and catalase (CAT) activities, with the same change trend as their expression levels. Therefore, GhSAMDC1 increased the contents of Spd and Spm of transgenic plants by increasing the expression of genes related to Spd and Spm synthesis under salt stress, Spd and Spm directly or indirectly increased the activity of enzymes related to antioxidant system, and enhanced the salt tolerance of Arabidopsis thaliana by scavenging H2O2 and other reactive oxygen species.

Key words: Arabidopsis thaliana, cotton S-adenosylmethionine decarboxylase gene, salt stress, antioxidant enzyme

Fig. 1

Phylogenetic analysis of homologous proteins of upland cotton GhSAMDC1 and other species and identification of transgenic Arabidopsis thaliana A: phylogenetic analysis of homologous proteins of upland cotton GhSAMDC1 and other species; B: expression vector of GhSDAMC1; C: DNA identification of transgenic Arabidopsis thaliana (GhSAMDC1), M, 1, 2, 3, 4, and 5 were marker, transgenic line 1-4, transgenic line 1-12, transgenic line 1-14, positive control, and negative control, respectively, the size of the target gene fragment was 1035 bp; D: expression analysis of GhSAMDC1 in Arabidopsis thaliana."

Fig. 2

Effect of overexpression of GhSAMDC1 on endogenous polyamines contents in Arabidopsis thaliana A-D: total endogenous polyamines, Put, Spd, and Spm contents in wild type and transgenic lines. The contents of endogenous polyamines in Arabidopsis thaliana leaves were determined by high performance liquid chromatography (HPLC) after normal cultured for 30 days, and Duncan method was used to test the difference significance (* P < 0.05, ** P < 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment."

Supplementary Fig. 1

Expression analysis of GhSAMDC1 in cotton under 300 mmol L-1 NaCl treatment 0, 1, 3, 6, 12, 24, 48, and72 represent cotton seedlings (YZ-1) after normal culture for 30 days treated with 300 mmol L-1 NaCl for 0, 1, 3, 6, 12, 24, 48, and 72 hours, respectively. Samples were taken at 0, 1, 3, 6, 12, 24, 48, and 72 hours of treatments to detect the relative expression of GhSAMDC1 in different treatment periods. The above experiments were repeated three times biologically and three times technically in each experiment."

Fig. 3

Effect of overexpression of GhSAMDC1 on salt tolerance in Arabidopsis thaliana A: schematic diagram of seed distribution; B-C: phenotypes of wild type and transgenic Arabidopsis thaliana cultivated under normal and salt stress for 15 days; D-E: fresh weight and leaf number of wild type and transgenic Arabidopsis thaliana after normal culture for 15 days; F-H: survival rate, fresh weight and leaf number of wild type and transgenic Arabidopsis thaliana after 100 mmol L-1 NaCl treatment for 15 days. Duncan method was used to test the difference significance (* P < 0.05, ** P < 0.01), the results were represented by mean (±standard), the above experiments were repeated three times biologically and three times technically in each experiment."

Fig. 4

Effects of overexpression of GhSAMDC1 on chlorophyll contents in Arabidopsis thaliana leaves under salt stress A-C: chlorophyll content of wild type and transgenic lines under salt stress. The leaves of Arabidopsis thaliana were selected to detect the content of chlorophyll a and b after treated with 100 mmol L-1 NaCl for 15 days. Duncan method was used to test the difference significance (*P < 0.05, **P < 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment."

Fig. 5

Effects of overexpression of GhSAMDC1 on endogenous polyamine content in Arabidopsis thaliana under salt stress A-D: comparison of total endogenous polyamines, Put, Spd, and Spm contents between wild type and transgenic lines under normal and salt stress conditions. The contents of endogenous polyamines in Arabidopsis thaliana leaves were determined by high performance liquid chromatography (HPLC) after normal culture and treatment with 100 mmol L-1 NaCl for 15 days. Duncan’s method was used to test the difference significance (* P < 0.05, ** P < 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment."

Fig. 6

Effects of overexpression of GhSAMDC1 on endogenous gene expression in Arabidopsis thaliana under salt stress A-C: relative expression of AtSPDS1, AtSPDS2, and AtSPMS in wild type and transgenic Arabidopsis thaliana. The contents of endogenous polyamines in Arabidopsis thaliana leaves were determined by high performance liquid chromatography (HPLC) after treated with 100 mmol L-1 NaCl for 15 days. Duncan’s method was used to test the difference significance (* P < 0.05, ** P < 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment."

Fig. 7

Effects of overexpression of GhSAMDC1 on contents of H2O2, MDA, and ion permeability in Arabidopsis thaliana leaves under salt stress A: DAB staining of wild type and transgenic lines under salt stress; B-D: contents of H2O2, MDA and ion permeability of wild type and transgenic lines under salt stress. The leaves of Arabidopsis thaliana were stained with DAB, and the contents of H2O2, MDA and ion permeability were measured after treated with 100 mmol L-1 NaCl for 15 days. Duncan’s method was used to test the difference significance (* P < 0.05, ** P < 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment."

Fig. 8

Effects of overexpression of GhSAMDC1 on activity and expression of antioxidant enzymes in Arabidopsis thaliana under salt stress A-C: enzyme activity of CAT, SOD, and POD in wild type and transgenic lines under salt stress; D-F: analysis of CAT, SOD, and POD expression in wild type and transgenic lines under salt stress. The leaves of Arabidopsis thaliana were selected to detect the activity and relative expression of CAT, SOD, and POD after treated with 100 mmol L-1 NaCl for 15 days, respectively. Duncan’s method was used to test the difference significance (* P < 0.05, ** P < 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologic ally and three times technically in each experiment."

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