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. 1993 Dec;103(4):1155–1163. doi: 10.1104/pp.103.4.1155

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.).

B D McKersie 1, Y Chen 1, M de Beus 1, S R Bowley 1, C Bowler 1, D Inzé 1, K D'Halluin 1, J Botterman 1
PMCID: PMC159101  PMID: 8290627

Abstract

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.

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Selected References

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  1. Bannister J. V., Bannister W. H., Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem. 1987;22(2):111–180. doi: 10.3109/10409238709083738. [DOI] [PubMed] [Google Scholar]
  2. Bowler C., Slooten L., Vandenbranden S., De Rycke R., Botterman J., Sybesma C., Van Montagu M., Inzé D. Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J. 1991 Jul;10(7):1723–1732. doi: 10.1002/j.1460-2075.1991.tb07696.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Bridges S. M., Salin M. L. Distribution of iron-containing superoxide dismutase in vascular plants. Plant Physiol. 1981 Aug;68(2):275–278. doi: 10.1104/pp.68.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dean C., Jones J., Favreau M., Dunsmuir P., Bedbrook J. Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Res. 1988 Oct 11;16(19):9267–9283. doi: 10.1093/nar/16.19.9267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Edwards K., Johnstone C., Thompson C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 1991 Mar 25;19(6):1349–1349. doi: 10.1093/nar/19.6.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giannopolitis C. N., Ries S. K. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol. 1977 Feb;59(2):309–314. doi: 10.1104/pp.59.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gupta A. S., Heinen J. L., Holaday A. S., Burke J. J., Allen R. D. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1629–1633. doi: 10.1073/pnas.90.4.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Herrera-Estrella L., Van den Broeck G., Maenhaut R., Van Montagu M., Schell J., Timko M., Cashmore A. Light-inducible and chloroplast-associated expression of a chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature. 1984 Jul 12;310(5973):115–120. doi: 10.1038/310115a0. [DOI] [PubMed] [Google Scholar]
  10. Jacobs J. M., Jacobs N. J., Sherman T. D., Duke S. O. Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley. Plant Physiol. 1991 Sep;97(1):197–203. doi: 10.1104/pp.97.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Odell J. T., Nagy F., Chua N. H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. 1985 Feb 28-Mar 6Nature. 313(6005):810–812. doi: 10.1038/313810a0. [DOI] [PubMed] [Google Scholar]
  12. Pitcher L. H., Brennan E., Hurley A., Dunsmuir P., Tepperman J. M., Zilinskas B. A. Overproduction of petunia chloroplastic copper/zinc superoxide dismutase does not confer ozone tolerance in transgenic tobacco. Plant Physiol. 1991 Sep;97(1):452–455. doi: 10.1104/pp.97.1.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Senaratna T., McKersie B. D., Stinson R. H. Simulation of dehydration injury to membranes from soybean axes by free radicals. Plant Physiol. 1985 Feb;77(2):472–474. doi: 10.1104/pp.77.2.472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Spychalla J. P., Desborough S. L. Superoxide Dismutase, Catalase, and alpha-Tocopherol Content of Stored Potato Tubers. Plant Physiol. 1990 Nov;94(3):1214–1218. doi: 10.1104/pp.94.3.1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tepperman J. M., Dunsmuir P. Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity. Plant Mol Biol. 1990 Apr;14(4):501–511. doi: 10.1007/BF00027496. [DOI] [PubMed] [Google Scholar]
  16. Vantoai T. T., Bolles C. S. Postanoxic Injury in Soybean (Glycine max) Seedlings. Plant Physiol. 1991 Oct;97(2):588–592. doi: 10.1104/pp.97.2.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wise R. R., Naylor A. W. Chilling-enhanced photooxidation : evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. Plant Physiol. 1987 Feb;83(2):278–282. doi: 10.1104/pp.83.2.278. [DOI] [PMC free article] [PubMed] [Google Scholar]

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