Skip to main content
SpringerLink
Log in

Two soybean seed lipoxygenase nulls accumulate reduced levels of lipoxygenase transcripts

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Summary

Soybean (Glycine max L. [Merrill]) seed lipoxygenase cDNA clones were recovered from two cDNA libraries: a size-selected library in pBR322 and an expression library in pUC8. The pUC8 library was made with total poly(A)+ embryo RNA and was screened with antiserum to lipoxygenase-1, one of 3 seed lipoxygenase isozymes. Three lipoxygenase antigen-producing clones were identified: two with identical cDNA inserts of 977 nucleotides representing an open-reading frame and a third truncated clone bearing a 3′ end common to the longer clones. A long clone, pAL-134, was chosen for further study and was used to screen the size-selected cDNA library from which sixteen clones were identified. They fall into two homology classes represented by pLX-10 (ca. 1360 bp) and pLX-65 (2047 bp).

The lipoxygenase expression clone pAL-134 hybridized much more strongly to pLX-65 than to pLX-10. pAL-134 and pLX-65 share 89% nucleotide homology and 75% deduced amino acid homology along their common sequence. Their deduced amino acid sequences each show 80% homology to sequences determined for isolated peptides of the lipoxygenase-1 isozyme.

pAL-134 hybridizes poorly with a 3.8 kb RNA from LOX-1 null (lx1) embryos while pLX-65 hybridizes more strongly, but still to a lesser extent than its hybridization to standard embryo RNA or to RNA from embryos lacking lipoxygenase-2 (lx2) or lipoxygenase-3 (lx3) protein.

The lx3 null lacks almost all embryo 3.8 kb RNA homologous to pLX-10. This hybridization pattern suggests that pLX-10 encodes LOX-3. Thus, the lx1 and lx3 genotypes accumulate little, if any, mRNA for the lipoxygenase-1 and lipoxygenase-3 isozymes, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Alwine JC, Kemp DJ, Parker BA, Reiser J, Renart J, Stark GR, Wahl GM: Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Meth Enzymol 68:220–242, 1979.

    Google Scholar 

  2. Axelrod B: Lipoxygenases. Whitaker JR (ed). Adv Chem Ser. Food Related Enzymes. 136:324–348, 1974.

  3. Axelrod B, Cheesbrough TM, Laakso S: Lipoxygenase from soybean. Meth Enzymol 71:441–451, 1981.

    Google Scholar 

  4. Bailey JM, Davidson N: Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem 70:75–85, 1976.

    Google Scholar 

  5. Barton KA, Thompson JF, Madison JT, Rosenthal R, Jarvis NP, Beachy RN: The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits. J Biol Chem 257:6089–6095, 1982.

    Google Scholar 

  6. Beachy RN, Thompson JF, Madison JT: Isolation of polyribosomes and messenger RNA acitve in in vitro synthesis of soybean seed proteins. Plant Physiol 61:139–144, 1978.

    Google Scholar 

  7. Birnboim HC: A rapid alkaline extraction method for the isolation of plasmid DNA. Meth Enzymol 17:243–255, 1979.

    Google Scholar 

  8. Bonner WM, Laskey RA: A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamid gels. Eur J Biochem 46:83–88, 1974.

    Google Scholar 

  9. Bousquet J-F, Thimann KV: Lipid peroxidation forms ethylene from 1-aminocyclopropane-1-carboxylic acid and may operate in leaf senescence. Proc Natl Acad Sci USA 81:1724–1727, 1984.

    Google Scholar 

  10. Chan HW-S: Soya-bean lipoxygenase: An iron-containing dioxygenase. Biochem Biophys Acta 327:32–35, 1973.

    Google Scholar 

  11. Christopher JP, Pistorius EK, Axelrod B: Isolation of an isozyme of soybean lipoxygenase. Biochim Biophys Acta 198:12–19, 1970.

    Google Scholar 

  12. Christopher JP, Pistorius EK, Axelrod B: Isolation of a third isoenzyme of soybean lipoxygenase. Biochim Biophys Acta 284:54–62, 1970.

    Google Scholar 

  13. Cohen SN, Chang ACY, Hsu L: Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114, 1973.

    Google Scholar 

  14. Goldberg RB, Hoschek G, Tam SH, Ditta GS, Breidenbach RW: Abundance, diversity, and regulation of mRNA sequence sets in soybean embryogenesis. Dev Biol 83:201–217, 1981.

    Google Scholar 

  15. Grunstein M, Hogness DS: Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965, 1975.

    Google Scholar 

  16. Guss PL, Richardson T, Stahmann MA: The oxidation-reduction enzymes of wheat. III. Isoenzymes of lipoxidase in wheat fractions and soybean. Cereal Chem 44:607–610, 1967.

    Google Scholar 

  17. Guss PL, Richardson T, Stahmann MA: Oxidation of various lipid substrates with unfractionated soybean and wheat lipoxydase. J Am Oil Chem Soc 45:272–276, 1968.

    Google Scholar 

  18. Haas LW, Bohn RM: Bleaching bread dough. US Patent 1957 333 (to JR Short Milling Company), 1934.

  19. Hanahan D: Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580, 1983.

    Google Scholar 

  20. Hanahan D, Meselsohn M: A protocol for high density screening of plasmids in Chi 1776. Gene 10:63–67, 1980.

    Google Scholar 

  21. Harman GE, Mattick LR: Association of lipid oxidation with seed ageing and death. Nature 260:323–324, 1976.

    Google Scholar 

  22. Helfman DM, Feramisco JR, Fiddes JC, Thomas GP, Hughes SH: Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci USA 80:31–35, 1983.

    Google Scholar 

  23. Hildebrand DF, Hymowitz T: Two soybean genotypes lacking lipoxygenase-1. J Am Oil Chem Soc 58:583–586, 1981.

    Google Scholar 

  24. Hildebrand DF, Hymowitz T: Inheritance of lipoxygenase-1 activity in soybean seeds. Crop Sci 22:851–853, 1982.

    Google Scholar 

  25. Hildebrand DF, Hymowitz T: Carotene and chlorophyll bleaching by soybeans with and without seed lipoxygenase-1. J Agric Food Chem 30:705–708, 1982.

    Google Scholar 

  26. Holman RT: Lipoxidase activity and fat composition of germinating soybeans. Arch Biochem Biophys 17:459–466, 1948.

    Google Scholar 

  27. Ikediobi CO, Snyder HE: Cooxidation of β-carotene by an isoenzyme of soybean lipoxygenase. J Agric Food Chem 25:124–127, 1977.

    Google Scholar 

  28. Kinsella JE: Functional properties of soy proteins. J Am Oil Chem Soc 56:242–258, 1977.

    Google Scholar 

  29. Kitamura K, Davis CS, Kaizuma N, Nielsen NC: Genetic analysis of a null allele for lipoxygenase-3 in soybean seeds. Crop Sci 23:924–926, 1983.

    Google Scholar 

  30. Kitamura K: Biochemical characterization of lipoxygenase lacking mutants, L-1-less, L-2-less, and L-3-less soybeans. Agric Biol Chem 48:2339–2346, 1984.

    Google Scholar 

  31. Koch RB, Stern B, Ferrari CG: Linoleic acid and trilinolein as substrates for soybean lipoxydase(s). Arch Biochem Biophys 78:165–179, 1958.

    Google Scholar 

  32. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685, 1970.

    Google Scholar 

  33. Laskey RA, Mills AD: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335–341, 1975.

    Google Scholar 

  34. Maxam AM, Gilbert W: Sequencing end-labeled DNA with base-specific chemical cleavages. Meth in Enzymol 65:499–560, 1980.

    Google Scholar 

  35. Messing J, Vieira J: A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276, 1982.

    Google Scholar 

  36. Murray MG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325, 1980.

    Google Scholar 

  37. Parrish DJ, Leopold AC: On the mechanism of aging in soybean seeds. Plant Physiol 61:365–368, 1978.

    Google Scholar 

  38. Paterson BM, Roberts BE, Kuff EL: Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci USA 74:4370–4374, 1977.

    Google Scholar 

  39. Pinsky A, Grossman S, Trop M: Lipoxygenase content and antioxidant activity of some fruits and vegetables. J Food Sci 36:571–572, 1971.

    Google Scholar 

  40. Pistorius EK, Axelrod B: Iron, an essential component of lipoxygenase. J Biol Chem 249:3183–3186, 1974.

    Google Scholar 

  41. Polacco JC, SparksJr RB: Patterns of urease synthesis in developing soybeans. Plant Physiol 70:189–194, 1982.

    Google Scholar 

  42. Privett OS, Dougherty KA, Erdahl WL, Stolywho A: Studies on the lipid composition of developing soybeans. J Am Oil Chem Soc 50:516–520, 1973.

    Google Scholar 

  43. Rackis JJ, Sessa DJ, Hornig DH: Flavor problems of vegetable food proteins. J Am Oil Chem Soc 56:262–271, 1979.

    Google Scholar 

  44. Rigby PWJ, Dieckmann M, Rhodes C, Berg P: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251, 1977.

    Google Scholar 

  45. Roza M, Francke A: Soybean lipoxygenase: An ironcontaining enzyme. Biochim Biophys Acta 327:24–31, 1973.

    Google Scholar 

  46. Salzmann U, Kuhn H, Schewe T, Rapoport SM: Pentane formation during the anaerobic reactions of reticulocyte lipoxygenase: Comparison with lipoxygenase from soybeans and green pea seeds. Biochim Biophys Acta 795:535–542, 1984.

    Google Scholar 

  47. Sammuelson B: Mediators of immediate hypersensitivity reactions and inflammation. Science 220:568–575, 1983.

    Google Scholar 

  48. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467, 1977.

    Google Scholar 

  49. Schuler MA, Ladin BF, Polacco JC, Freyer GA, Beachy RN: Structural sequences are conserved in the genes coding for the α, α′ and β subunits of the soybean 7S seed storage protein. Nucl Acids Res 10:8245–8261, 1982.

    Google Scholar 

  50. Schuler MA, Schmitt ES, Beachy RN: Closely related families of genes code for the α and α′ subunits of the soybean 7S storage protein complex. Nuc Acids Res 10:8225–8244, 1982.

    Google Scholar 

  51. Shukle RH, Mundock LL: Lipoxygenase, trypsin inhibitor and lectin tromsoybeans: effects on larval growth of Manduca sexta. Environ Entomol 12:789–791, 1983.

    Google Scholar 

  52. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517, 1975.

    Google Scholar 

  53. Takeo T, Tsushida T: Changes in lipoxygenase activity in relation to lipid degradation in plucked tea shoots. Phytochemistry 19:2521–2522, 1980.

    Google Scholar 

  54. Takishima K, Mamiya G, Tsunijiro S: Partial amino acid sequence of Jack bean urease. J Natl Def Coll 5:19–23, 1980.

    Google Scholar 

  55. Theorell H, Holman RT, Akeson A: Crystalline lipoxidase. Acta Chem Scand 1:571–576, 1947.

    Google Scholar 

  56. Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201–5205, 1980.

    Google Scholar 

  57. Towbin H, Staehlin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354, 1979.

    Google Scholar 

  58. Veldink GA, Vliegenthart JFG: Lipoxygenases, nonheme iron-containing enzymes pp 139–161. In: Adv in Inorganic Biochem Vol VI, Elsevier, NY, 1985.

    Google Scholar 

  59. Vernooy-Gerritsen M, Bos ALM, Veldink GA, Vliegenthart JFG: Localization of lipoxygenases-1 and-2 in germinating soybean seeds by an indirect immunofluorescence technique. Plant Physiol 73:262–267, 1983.

    Google Scholar 

  60. Vernooy-Gerritsen M, Leunissen JLM, Veldink GA, Vliegenthart JFG: Intracellular localization of lipoxygenases-1 and-2 in germinating soybean seeds by indirect labeling with protein A-colloidal gold complexes. Plant Physiol 76:1070–1079, 1984.

    Google Scholar 

  61. Vieira J, Messing J: The pUC plasmids, and M13 mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268, 1982.

    Google Scholar 

  62. Zimmerman GL, Snyder HE: Role of calcium in activating soybean lipoxygenase-2. J Agric Food Chem 22:802–805, 1974.

    Google Scholar 

Download references

Author information

Author notes

  1. William G. Start

    Present address: Central Research, Plant Genetics, Pfizer, Inc., 06340, Groton, CT, U.S.A.

  2. Greg A. Freyer

    Present address: Department of Molecular Biology, Memorial Sloan Kettering Cancer Institute, 10021, New York, NY, U.S.A.

Authors and Affiliations

  1. Biochemistry Department, University of Missouri, 65212, Columbia, MO, U.S.A.

    William G. Start, Joseph C. Polacco & Greg A. Freyer

  2. Advanced Research Division, Agrigenetics Corp., 53716, Madison, WI, U.S.A.

    Yu Ma

  3. Department of Agronomy, University of Kentucky, 40546-0091, Lexington, KY, U.S.A.

    David F. Hildebrand & Mitchell Altschuler

Authors
  1. William G. Start
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph C. Polacco
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David F. Hildebrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Greg A. Freyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mitchell Altschuler
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Start, W.G., Ma, Y., Polacco, J.C. et al. Two soybean seed lipoxygenase nulls accumulate reduced levels of lipoxygenase transcripts. Plant Mol Biol 7, 11–23 (1986). https://doi.org/10.1007/BF00020127

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020127

Keywords

Search

Navigation