Plant Molecular Biology

, Volume 30, Issue 2, pp 229–241 | Cite as

Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley

  • Brian K. Tibbot
  • Ronald W. Skadsen
Research article


A putative α-glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A3 (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal α-glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI α-amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this α-glucosidase gene and two additional sequences that may be related genes or pseudogenes.

Key words

α-amylase barley GA gene-expression α-glucosidase maltase 


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  1. 1.
    Belanger FC, Brodl MR, Ho T-H D: Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells. Plant Physiol 83: 1354–1358 (1986).Google Scholar
  2. 2.
    Benton WD, Davis RW: Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182 (1977).Google Scholar
  3. 3.
    Blish MT, Sandstedt RM, Mecham DK: Action of wheat amylases on raw wheat starch. Cereal Chem 14: 605–628 (1937).Google Scholar
  4. 4.
    Chantret I, Lacasa M, Chevalier G, Ruf J, Islam I, Mantei N, Edwards Y, Swallow D, Rousset M: Sequence of the complete cDNA and the 5′ structure of the human sucrase-isomaltase gene. Possible homology with a yeast glucoamylase. Biochem J 285: 915–923 (1992).Google Scholar
  5. 5.
    Chrispeels MJ, Varner JE: Gibberellic acid-enhanced synthesis and release of α-amylase and ribonuclease by isolated aleurone layers. Plant Physiol 42: 398–406 (1967).Google Scholar
  6. 6.
    Clutterbuck VJ, Briggs DE: Enzyme formation and release by isolated barley aleurone layers. Phytochemistry 12: 537–546 (1973).Google Scholar
  7. 7.
    Coutinho PM, Reilly PJ: Structure-function relationships in the catalytic and starch binding domains of glucoamylase. Protein Engng 7: 393–400 (1994).Google Scholar
  8. 8.
    Dohmen RJ, Strasser AWM, Dahlems UM, Hollenberg CP: Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in Saccharomyces cerevisiae. Gene 95: 111–121 (1990).Google Scholar
  9. 9.
    Dunn G: A model for starch breakdown in higher plants. Phytochemistry 13: 1341–1346 (1974).Google Scholar
  10. 10.
    Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specificity. Anal Biochem 132: 6–13 (1983).Google Scholar
  11. 11.
    Fincher GB, Lock PA, Morgan MM, Lingelbach K, Wettenhall REH, Mercer JFB, Brandt A, Thomsen KK: Primary structure of the (1–3, 1–4)-β-D-glucan 4-glucohydrolase from barley aleurone. Proc Natl Acad Sci USA 83: 2081–2085 (1986).Google Scholar
  12. 12.
    Geber A, Williamson PR, Rex JH, Sweeney EC, Bennett JE: Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization. J Bact 174: 6992–6996 (1992).Google Scholar
  13. 13.
    Genetics Computer Group, University of Wisconsin, Sequence Analysis Software, Wisconsin Package, Version 8. 1 (1994).Google Scholar
  14. 14.
    Green F, Edwards Y, Hauri H-P, Povey S, Ho MW, Pinto M, Swallow D: Isolation of a cDNA probe for a human jejunal brush-border hydrolase, sucrase-isomaltase, and assignment of the gene locus to chromosome 3. Gene 57: 101–110 (1987).Google Scholar
  15. 15.
    Hardie DG: Control of carbohydrase formation by gibberellic acid in barley endosperms. Phytochemistry 14: 1719–1722 (1975).Google Scholar
  16. 16.
    Hata Y, Tsuchiya K, Kitamoto K, Gomi K, Kumagai C, Tamura G, Hara S: Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae. Gene 108: 145–150 (1991).Google Scholar
  17. 17.
    Henrissat B, Bairoch A: New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293: 781–788 (1993).Google Scholar
  18. 18.
    Hermans MMP, Kroos MA, van Beeumen J, Oostra BA, Reuser AJJ: Human lysosomal α-glucosidase. Characterization of the catalytic site. J Biol Chem 266: 13507–13512 (1991).Google Scholar
  19. 19.
    Hoefsloot LH, Hoogeveen-Westerveld M, Kroos MA, Van Beeuman J, Reuser AJJ, Oostra BA: Primary structure and processing of lysosomal α-glucosidase; homology with the intestinal sucrase-isomaltase complex. EMBO J 7: 1697–1704 (1988).Google Scholar
  20. 20.
    Hong SH, Marmur J: Primary structure of the maltase gene of the Mal 6 locus of Saccharomyces carlsbergensis. Gene 41: 75–84 (1984).Google Scholar
  21. 21.
    Hostinova E, Balanova J, Gasperik J: The nucleotide sequence of the glucoamylase gene GLA1 from Saccharomypsis fibuligera KZ. FEMS Microbiol Lett 83: 103–108 (1991).Google Scholar
  22. 22.
    Hunziker W, Spiess M, Semenza G, Lodish HF: The sucrase-isomaltase complex: Primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein. Cell 46: 227–234 (1986).Google Scholar
  23. 23.
    James AA, Blackmer K, Racioppi JV: A salivary gland-specific maltase-like gene of the vector mosquito, Aedes aegypti. Gene 75: 73–83 (1989).Google Scholar
  24. 24.
    Janse BJH, Steyn AJC, Pretorius IS: Regional sequence homologies in starch-degrading enzymes. Curr Genet 24: 400–407 (1993).Google Scholar
  25. 25.
    Jespersen HM, MacGregor EA, Henrissat B, Sierks MR, Svensson B: Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes. J Prot Chem 12: 791–805 (1993).Google Scholar
  26. 26.
    Jorgensen BB, Jorgensen OB: Barley malt α-glucosidase with isomaltase activity. Acta Chem Scand 17: 1765–1770 (1963).Google Scholar
  27. 27.
    Jorgensen OB: Barley malt α-glucosidase. VI. Localization and development during barley germination. Acta Chem Scand 19: 1014–1015 (1965).Google Scholar
  28. 28.
    Kinsella BT, Hogan S, Larkin A, Cantwell BA: Primary structure and processing of the Candida tsukubaensis α-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal α-glucosidase. Eur J Biochem 202: 657–664 (1991).Google Scholar
  29. 29.
    Kislev N, Rubenstein I: Utility of ethidium bromide in the extraction from whole plants of high molecular weight maize DNA. Plant Physiol 66: 1140–1143 (1980).Google Scholar
  30. 30.
    Konishi Y, Okamoto A, Takahashi J, Aitani M, Nakatani N: Effects of Bay m 1099, an α-glucosidase inhibitor, on starch metabolism in germinating wheat seeds. Biosci Biotechnol Biochem 58: 135–139 (1994).Google Scholar
  31. 31.
    Koehler SM, Ho T-H D: Hormonal regulation, processing and secretion of cysteine proteinases in barley aleurone layers. Plant Cell 2: 769–783 (1990).Google Scholar
  32. 32.
    Kopetzki E, Buckel P, Schumacher G: Cloning and characterization of Baker's yeast α-glucosidase: over-expression in a yeast strain devoid of vacuolar proteinases. Yeast 5: 11–24 (1989).Google Scholar
  33. 33.
    Kozak M: Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292 (1986).Google Scholar
  34. 34.
    Kreis M, Williamson M, Buxton B, Pywell J, Hejgaard J, Svendsen I: Primary structure and differential expression of β-amylase in normal and mutant barleys. Eur J Biochem 169: 517–525 (1987).Google Scholar
  35. 35.
    MacGregor AW, Lenoir C: Studies on α-glucosidase in barley malt. J Inst Brew 93: 334–337 (1987).Google Scholar
  36. 36.
    Martiniuk F, Mehler M, Tzall S, Meredith G, Hirschhorn R: Sequence of the cDNA and 5′-flanking region for human acid α-glucosidase, detection of an intron in the 5′ untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences. DNA Cell Biol 9: 85–94 (1990).Google Scholar
  37. 37.
    Naim HY, Niermann T, Kleinhans U, Hollenberg CP, Strasser AWM: Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal α-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene. FEBS Lett 294: 109–112 (1991).Google Scholar
  38. 38.
    Nakao M, Nakayama T, Kakudo A, Inohara M, Harada M, Omura F, Shibano Y: Structure and expression of a gene coding for thermostable α-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606. Eur J Biochem 220: 293–300 (1994).Google Scholar
  39. 39.
    Rave N, Crkvenjakov R, Boedtker H: Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucl Acids Res 6: 3559–3567 (1979).Google Scholar
  40. 40.
    Rick W, Stegbauer HP: Measurement of reducing groups. In: Bergmeyer HU (ed) Methods of Enzymatic Analysis, 2nd ed., vol 2, pp. 885–889. Academic Press, New York (1974).Google Scholar
  41. 41.
    Rogers JC: Two barley α-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260: 3731–3738 (1985).Google Scholar
  42. 42.
    Rogers JC, Milliman C: Isolation and sequence analysis of a barley alpha-amylase cDNA clone. J Biol Chem 258: 8169–8174 (1983).Google Scholar
  43. 43.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed., pp. 1.42–1.46, 9.38–9.40. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  44. 44.
    Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).Google Scholar
  45. 45.
    Sierks MR, Ford C, Reilly PJ, Svensson B: Functional roles and subsite locations of Leu 177, Trp 178 and Asn 182 of Aspergillus awamori glucoamylase determined by sitedirected mutagenesis. Protein Engng 6: 75–79 (1993).Google Scholar
  46. 46.
    Skadsen RW: Aleurones from a barley with low α-amylase activity become highly responsive to gibberellin when detached from the starchy endosperm. Plant Physiol 102: 195–203 (1993).Google Scholar
  47. 47.
    Skadsen RW, Tibbot BK: Temporal expression patterns of alpha-amylase isozymal genes in polysomal and total RNAs of germinating barleys. J Cereal Sci 19: 199–208 (1994).Google Scholar
  48. 48.
    Snyder M, Davidson N: Two gene families clustered in a small region of the Drosophila genome. J Mol Biol 166: 101–118 (1983).Google Scholar
  49. 49.
    Stark JR, Yin XS: Evidence for the presence of maltase and α-glucosidase isozymes in barley. J Inst Brew 93: 108–112 (1987).Google Scholar
  50. 50.
    Sun Z, Henson CA: A quantitative assessment of the importance of barley seed α-amylase, β-amylase, debranching enzyme, and α-glucosidase in starch degradation. Arch Biochem Biophys 284: 298–305 (1991).Google Scholar
  51. 51.
    Sun Z, Henson CA: Degradation of native starch granules by barley α-glucosidases. Plant Physiol 94: 320–327 (1990).Google Scholar
  52. 52.
    Svensson B: Regional distant sequence homology between amylases, α-glucosidases and transglucanosylases. FEBS Lett 230: 72–76 (1988).Google Scholar
  53. 53.
    Tanaka Y, Ashikari T, Nakamura N, Kiuchi N, Shibano Y, Amachi T, Yoshizumo H: Comparison of amino acid sequence of three glucoamylases and their structure-function relationships. Agric Biol Chem 50: 965–969 (1986).Google Scholar
  54. 54.
    Tapio S, Yeh F, Shuman HA, Boos W: The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase. J Biol Chem 266: 19450–19458 (1991).Google Scholar
  55. 55.
    Thomas PS: Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Meth Enzymol 100: 255–266 (1983).Google Scholar
  56. 56.
    Watson TG, Novellie L: Extraction of Sorghum vulgare and Hordeum vulgare α-glucosidase. Phytochemistry 13: 1037–1041 (1974).Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Brian K. Tibbot
    • 1
    • 2
  • Ronald W. Skadsen
    • 1
    • 2
  1. 1.Department of AgronomyUniversity of WisconsinMadisonUSA
  2. 2.Cereal Crops Research UnitUSDA, Agricultural Research ServiceMadisonUSA

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