Advertisement

Planta

, Volume 223, Issue 5, pp 1010–1023 | Cite as

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus

  • Kirsten A. Nielsen
  • Maria Hrmova
  • Janni Nyvang Nielsen
  • Karin Forslund
  • Stefan Ebert
  • Carl E. Olsen
  • Geoffrey B. Fincher
  • Birger Lindberg MøllerEmail author
Original Article

Abstract

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic β-d-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing β-d-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic β-d-glucosidase from sorghum. This resulted in a 35–60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-β-d-glucan exohydrolase isozyme βII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-β-d-glucan exohydrolase isozyme βII was found to hydrolyze the cyanogenic β-d-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.

Keywords

Cyanogenesis 

Abbreviations

Glc

Glucose

GFP

Green fluorescent

Notes

Acknowledgements

We thank Mogens Houmøller for generously providing the B. graminis f. sp. Hordei isolate. Professor Asim Esen, Virginia Polytechnic Institute and State University Blacksburg, VA, USA is thanked for helpful discussions. This work was supported by grants from the Danish National Research Foundation to Center for Molecular Plant Physiology (PlaCe), from the Swedish Agricultural Research Council to KF, by a EU Marie Curie training grant to SE, and from the Australian Research Council and the Grains Research and Development Corporation to GBF.

References

  1. Bak S, Kahn RA, Nielsen HL, Møller BL, Halkier BA (1998) Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin. Plant Mol Biol 36:393–405CrossRefPubMedGoogle Scholar
  2. Barrett T, Suresh CG, Tolley SP, Dodson EJ, Hughes MA (1995) The crystal structure of cyanogenic β-glucosidase from white clover, a family 1 glycosyl hydrolase. Structure 3:951–960CrossRefPubMedGoogle Scholar
  3. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28:235–242CrossRefPubMedGoogle Scholar
  4. Boyle A, Perry-O’Keefe H (1994) Preparation and detection of digoxigenin-labelled DNA probes. In: Chanda VB (ed) Current protocols in molecular biology, vol 1, sections 3.18.5–3.18.6. John Wiley & Sons Inc, New York, USAGoogle Scholar
  5. Blundell T, Carney D, Gardner S, Hayes F, Howlin B, Hubbard T, Overington J, Singh DA, Sibanda BL, Sutcliffe M (1988) 18th Sir Hans Krebs lecture. Knowledge-based protein modelling and design. Eur J Biochem 172:513–520CrossRefPubMedGoogle Scholar
  6. Burmeister WP, Cottaz S, Rollin P, Vasella A, Henrissat B (2000) High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. J Biol Chem 275:39385–39393CrossRefPubMedGoogle Scholar
  7. Cicek M, Esen A (1998) Structure and expression of a dhurrinase (β-glucosidase) from sorghum. Plant Physiol 116:1469–1478CrossRefPubMedGoogle Scholar
  8. Cicek M, Blanchard D, Bevan DR, Esen A (2000) The aglycone specificity-determining sites are different in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (maize beta-glucosidase) and dhurrinase (sorghum β-glucosidase). J Biol Chem 275:20002–20011CrossRefPubMedGoogle Scholar
  9. Chothia A, Lesk AM (1986) The relation between the divergence of sequence and structure in proteins. EMBO J 5:823–826PubMedGoogle Scholar
  10. Clark M, Cramer RDI, Opdenbosch VD (1989) Validation of the general purpose Tripos 5.2 force field. J Comp Chem 8:982–1012CrossRefGoogle Scholar
  11. Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies G, Henrissat B, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12Google Scholar
  12. DeLano WL (2002) The PyMOL molecular graphics system; http://www.pymol.org
  13. Forslund K, Pettersson J, Ahmed E, Jonsson L (1998) Settling behaviour of Rhopalosiphum padi (L.) in relation to cyanogenic glycosides and gramine contents in barley. Acta Agr Scand B-S P 48:107–112Google Scholar
  14. Gay JL, Martin M, Ball E (1985) The impermeability of powdery mildew conidia and their germination in arid environments. Plant Pathol 34:353–362CrossRefGoogle Scholar
  15. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling. Electrophoresis 18:2714–2723CrossRefPubMedGoogle Scholar
  16. Guex N, Peitsch MC (1999) Molecular modelling of proteins. Immun News 6:132–134Google Scholar
  17. Halkier BA, Møller BL (1989) Biosynthesis of the cyanogenic glucoside dhurrin in seedlings of Sorghum bicolor (L.) Moench and partial purification of the enzyme system involved. Plant Physiol 90:1552–1559PubMedGoogle Scholar
  18. Henrissat B (1998) Glycosidase families. Biochem Soc Trans 26:153–156PubMedGoogle Scholar
  19. Hösel W, Tober I, Eklund SH, Conn EE (1987) Characterization of β-glucosidases with high specificity for the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench seedlings. Arch Biochem Biophys 252:152–162CrossRefPubMedGoogle Scholar
  20. Hrmova M, Fincher GB (1993) Purification and properties of three (1,3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare). Biochem J 289:453–461PubMedGoogle Scholar
  21. Hrmova M, Harvey AJ, Wang J, Shirley NJ, Jones GP, Stone BA, Høj PB, Fincher GB (1996) Barley β-d-glucan exohydrolases with β-d-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone. J Biol Chem 271:5277–5286CrossRefPubMedGoogle Scholar
  22. Hrmova M, MacGregor EA, Biely P, Stewart RJ, Fincher GB (1998) Substrate binding and catalytic mechanism of a barley β-d-glucosidase/(1,4)-β-d-glucan exohydrolase. J Biol Chem 273:11134–11143CrossRefPubMedGoogle Scholar
  23. Hrmova M, Fincher GB (2001) Structure-function relationships of β-d-glucan endo- and exohydrolases from higher plants. Plant Mol Biol 47:73–91CrossRefPubMedGoogle Scholar
  24. Hrmova M, De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, Fincher GB (2002) Structural basis for a broad specificity in higher plant β-d-glucan glucohydrolases. Plant Cell 14:1033–1052CrossRefPubMedGoogle Scholar
  25. Hughes MA (1991) The cyanogenic polymorphism in Trifolium repens L. (white clover). Heredity 66:105–115Google Scholar
  26. Hughes MA, Brown K, Murray BS, Oxtoby E, Hughes J (1992) A molecular and biochemical analysis of the structure of the cyanogenic β-glucosidase from cassava Manihot esculenta Crantz. Arch Biochem Biophys 295:273–279CrossRefPubMedGoogle Scholar
  27. Ibenthal W-D, Pourmohseni H, Grosskopf S, Oldenburg H, Shafiei-Azad S (1993) New approaches towards biochemical mechanisms of resistance/susceptibility of Gramineae to powdery mildew (Erysiphe graminis). Angew Bot 67:97–106Google Scholar
  28. Jones P, Andersen MD, Nielsen JS, Høj PB, Møller BL (2000) The biosynthesis, degradation, transport and possible function of cyanogenic glucosides. In: Romeo JT, Ibrahim R, Varin L, de Luca V (eds) Evolution of metabolic pathways. Elsevier, New York, pp 191–247Google Scholar
  29. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Cryst A47:110–119Google Scholar
  30. Kakes P (1985) Linamarase and other β-glucosidases are present in the cell walls of Trifolium repens L. leaves Planta 166:156–160CrossRefGoogle Scholar
  31. Kleywegt GJ, Jones TA (1998) Databases in protein crystallography. Acta Cryst D54:1119–1131Google Scholar
  32. Koch B, Nielsen VS, Halkier BA, Olsen CE, Møller BL (1992) The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantz). Arch Biochem Biophys 292:141–150CrossRefPubMedGoogle Scholar
  33. Kojima M, Poulton JE, Thayer SS, Conn EE (1979) Tissue distributions of dhurrin and of enzymes involved in its metabolism in leaves of Sorghum bicolor. Plant Physiol 63:1022–1028PubMedGoogle Scholar
  34. Kristensen BK, Ammitzbøll H, Rasmussen SK, Nielsen KA (2001) Transient expression of a vacuolar peroxidase increases susceptibility of epidermal barley cells to powdery mildew. Mol Plant Pathol 2:311–318CrossRefGoogle Scholar
  35. Leah R, Kigel J, Svendsen I, Mundy J (1995) Biochemical and molecular characterization of a barley seed β-glucosidase. J Biol Chem 270:15789–15797CrossRefPubMedGoogle Scholar
  36. Lechtenberg M, Nahrstedt A (1999) Cyanogenic glucosides. In: Ikan R (ed) Naturally occurring glucosides. John Wiley & Sons Ltd., Chicester, UK, pp 147–191Google Scholar
  37. Lieberei R (1986) Cyanogenesis of Hevea brasiliensis during infection with Microcyclus ulei. J Phytopathol 115:134–146Google Scholar
  38. Lieberei R, Biehl B, Giesemann A, Junqueira NTV (1989) Cyanogenesis inhibits active defense reactions in plants. Plant Physiol 90:33–36PubMedGoogle Scholar
  39. Lieberei R, Fock H, Biehl B (1996) Cyanogenesis inhibits active defense in plants: Inhibition by gaseous HCN of photosynthetic CO2 fixation and respiration in intact leaves. J Appl Bot 70:232–238Google Scholar
  40. Luthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356:83–85CrossRefPubMedGoogle Scholar
  41. Møller BL, Seigler DS (1999) Biosynthesis of cyanogenic glucosides, cyanolipids, and related compounds. In: Singh BK (ed) Plant amino acids, biochemistry and biotechnology. Marcel Dekker, New York, pp 563–609Google Scholar
  42. Nelson N (1944) A photometric adaptation of the Somogyi method for determination of glucose. J Biol Chem 153:375–380Google Scholar
  43. Nicolls A, Sharp K, Hönig B (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 4:281–296CrossRefGoogle Scholar
  44. Nielsen KA, Olsen O, Oliver RP (1999) A transient expression system to assay putative antifungal genes on powdery mildew infected barley leaves. Physiol Mol Plant Pathol 54:1–12CrossRefGoogle Scholar
  45. Nielsen KA, Nicholson RL, Carver TMV, Kunoh H, Oliver RP (2000) First touch: an immediate response to surface recognition in conidia of Blumeria graminis. Physiol Mol Plant Pathol 56:63–70CrossRefGoogle Scholar
  46. Nielsen KA, Olsen CE, Pontoppidan K, Møller BL (2002) Leucine-derived cyano glucosides in barley. Plant Physiol 129:1066–1075CrossRefPubMedGoogle Scholar
  47. Osbourn AE (1994) Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821–1831CrossRefGoogle Scholar
  48. Raabo E, Terkildsen TC (1960) On the enzymatic determination of blood glucose. Scand J Clin Lab Invest 12:402–407PubMedCrossRefGoogle Scholar
  49. Schulze-Lefert P, Vogel J (2000) Closing the ranks to attack by powdery mildew. Trends Plant Sci 5:343–348CrossRefPubMedGoogle Scholar
  50. Seigler D (1998) Cyanogenic glycosides and cyanolipids. In: Seigler D (ed) Plant secondary metabolism. Kluwer, Norwell, pp 273–299Google Scholar
  51. Selmar D (1993) Apoplastic occurrence of cyanogenic β-glucosidases and consequences for the metabolism of cyanogenic glucosides. In: Esen A (ed) β-Glucosidases: biochemistry and molecular biology. American Chemical Society, pp 191–204Google Scholar
  52. Selmar D, Lieberei R, Biehl B (1988) Mobilization and utilization of cyanogenic glucosides: the linustatin pathway. Plant Physiol 86:711–716PubMedCrossRefGoogle Scholar
  53. Selmar D, Lieberei R, Biehl B, Voigt J (1987) Hevea linamarase—a nonspecific β-glycosidase. Plant Physiol 83:557–563PubMedGoogle Scholar
  54. Shirasu K, Nielsen K, Piffanelli P, Oliver R, Schulze-Lefert P (1999) Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J 17:293–299CrossRefGoogle Scholar
  55. Simos G, Panagiotidis CA, Skoumbas A, Choli A, Ouzounis C, Georgatsos JG (1994) Barley β-glucosidase expression during seed germination and maturation and partial amino acid sequences. Biochem Biophys Acta 1199:52–57PubMedGoogle Scholar
  56. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23PubMedGoogle Scholar
  57. Swantson JS, Thomas WTB, Powell W, Young GR, Lawrence PE, Ramsey L, Waugh R (1999) Using molecular markers to determine barleys most suitable for malt whisky distilling. Mol Breed 5:103–109CrossRefGoogle Scholar
  58. Thayer SS, Conn EE (1981) Subcellular localization of dhurrin β-glucosidase and hydroxynitrile lyase in the mesophyll cells of shorghum leaf blades. Plant Physiol 67:617–622PubMedGoogle Scholar
  59. Till I (1987) Variability of expression of cyanogenesis in white clover (Trifolium reprens L.). Heredity 59:265–271Google Scholar
  60. Töpfer R, Matzeit V, Gronenborn B, Schell J, Steinbiss H (1987) A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acid Res 15:5890PubMedCrossRefGoogle Scholar
  61. Veibel S (1950) β-Glucosidase. In: Sumner JB, Myrbäck K (eds) The enzymes. Chemistry and mechanism of action, vol I. GEC Gad Publ., Copenhagen, pp 583–634Google Scholar
  62. Xu Z, Escamilla-Treviño LL, Zeng L, Lalgondar M, Bevan DR, Winkel BSJ, Mohammed A, Cheng C-L, Shih M-C, Poulton JE, Esen A (2004) Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1. Plant Mol Biol 55:343–367CrossRefPubMedGoogle Scholar
  63. Zagrobelny M, Bak S, Rasmussen AV, Jørgensen B, Naumann CM, Møller BL (2004) Cyanogenic glucosides and plant-insect interactions. Phytochemistry 65:293–306CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Kirsten A. Nielsen
    • 1
    • 2
    • 5
  • Maria Hrmova
    • 4
  • Janni Nyvang Nielsen
    • 1
  • Karin Forslund
    • 1
    • 6
  • Stefan Ebert
    • 1
  • Carl E. Olsen
    • 3
  • Geoffrey B. Fincher
    • 4
  • Birger Lindberg Møller
    • 1
    • 2
    Email author
  1. 1.Plant Biochemistry Laboratory, Department of Plant BiologyRoyal Veterinary and Agricultural UniversityFrederiksberg C, CopenhagenDenmark
  2. 2.Center for Molecular Plant PhysiologyRoyal Veterinary and Agricultural UniversityFrederiksberg C, CopenhagenDenmark
  3. 3.Department of Natural SciencesRoyal Veterinary and Agricultural UniversityFrederiksberg C, CopenhagenDenmark
  4. 4.School of Agriculture and Wine, and the Australian Centre for Plant Functional GenomicsUniversity of AdelaideGlen OsmondAustralia
  5. 5.Epilepsy HospitalDianalundDenmark
  6. 6.Department of Physiological Botany, EBCUppsala UniversityUppsalaSweden

Personalised recommendations