Plant Molecular Biology

, Volume 56, Issue 4, pp 661–669 | Cite as

Engineering cyanogen synthesis and turnover in cassava (Manihot esculenta)

  • Dimuth Siritunga
  • Richard Sayre


Cassava is the major root crop for a quarter billion subsistence farmers in sub-Saharan Africa. It is valued for its ability to grow in adverse environments and the food security it provides. Cassava contains potentially toxic levels of cyanogenic glycosides (linamarin) which protect the plant from herbivory and theft. The cyanogens, including linamarin and its deglycosylated product, acetone cyanohydrin, can be efficiently removed from the root by various processing procedures. Short-cuts in processing, which may occur during famines, can result in only partial removal of cyanogens. Residual cyanogens in cassava foods may cause neurological disorders or paralysis, particularly in nutritionally compromised individuals. To address this problem and to further understand the function of cyanogenic glycosides in cassava, we have generated transgenic cassava in which cyanogenic glycoside synthesis has been selectively inhibited in leaves and roots by antisense expression of CYP79D1/D2 gene fragments. The CYP79D1/D2 genes encode two highly similar cytochrome P450s that catalyze the first-dedicated step in cyanogenic glycoside synthesis. Transgenic plants in which the expression of these genes was selectively inhibited in leaves had substantially reduced (60– 94% reduction) linamarin leaf levels. Surprisingly, these plants also had a greater than a 99% reduction in root linamarin content. In contrast, transgenic plants in which the CYP79D1/D2 transcripts were reduced to non-detectable levels in roots had normal root linamarin levels. These results demonstrate that linamarin synthesized in leaves is transported to the roots and accounts for nearly all of the root linamarin content. Importantly, transgenic plants having reduced leaf and root linamarin content were unable to grow in the absence of reduced nitrogen (NH3) . Cassava roots have previously been demonstrated to have an active cyanide assimilation pathway leading to the synthesis of amino acids. We propose that cyanide derived from linamarin is a major source of reduced nitrogen for cassava root protein synthesis. Disruption of linamarin transport from leaves in CYP79D1/D2 anti-sense plants prevents the growth of cassava roots in the absence of an alternate source of reduced nitrogen. An alternative strategy for reducing cyanogen toxicity in cassava foods is to accelerate cyanogenesis and cyanide volatilization during food processing. To achieve this objective, we have expressed the leaf-specific enzyme hydroxynitrile lyase (HNL) in roots. HNL catalyzes the breakdown of acetone cyanohydrin to cyanide. Expression of HNL in roots accelerated cyanogenesis by more than three-fold substantially reducing the accumulation of acetone cyanohydrin during processing relative to wild-type roots.


cyanogenesis cyanogenic glycoside cytochrome P450 hydroxynitrile lyase linamarin 


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  1. Akintonwa, A., Tunwashe, O. 1992Fatal cyanide poisoning from cassava-based mealHuman Exp. Toxic.114749Google Scholar
  2. Andersen, M., Bush, P., Svendsen, I., Moller, B. 2000Cytochromes P450 from cassava catalyzing the first steps in the biosynthesis of the cyanogenic glycosides linamarin and lotaustralinJ. Biol. Chem.27519661975CrossRefPubMedGoogle Scholar
  3. Bak, S., Olsen, C.E., Halkier, B.A., Moller, B.L. 2002Transgenic tobacco and Arabidopsis plants expressing the two multifunctional sorghum cytochrome P450 enzymes, CYP79A1 and CYP71E1, are cyanogenic and accumulate metabolites derived from intermediates in Dhurrin biosynthesisPlant Physiol.12314371448CrossRefGoogle Scholar
  4. C. Balagopalan,G.Padmaja,S.Nanda,S.Morthy1988Cassava nutrition and toxicityIn: Cassava in FoodFeed and Industry. CRC Press, Boca Raton, FloridaGoogle Scholar
  5. M.Bediako, B.Tapper,G.Pritchard 1981Metabolism synthetic site and translocation of cyanogenic glucoside in cassavaIn: E. Terry (Ed.) Proceedings of the first triennial root crops symposium of the International society for tropical root crops IDRC Canada, pp. 143–148Google Scholar
  6. A.Bellotti,B.Arias,1993The possible role of HCN on the biology and feeding behavior of the cassava burrowing bug (Cyrtomenus bergi Froeschner)In: W.M. Roca and A.M. Thro (Eds.) Proceedings of the first international scientific meeting of the Cassava Biotechnology Network25–28 August 1992. Cali, Colombia: pp 406–409, Centro Internacional de Agricultura TropicalGoogle Scholar
  7. Bellotti, A., Riss, L. 1994Cassava cyanogenic potential and resistance to pests and diseasesActa Hort.375141151Google Scholar
  8. Best, R., Hargrove, T. 1994Cassava: the latest facts about an ancient cropCIAT PublicationCali, ColombiaGoogle Scholar
  9. Bokanga, M. 1994Processing of cassava leaves for human consumptionActa Hort.375203207Google Scholar
  10. Brusslan, J., Tobin, E. 1992Light-independent developmental regulation of cab gene expression in Arabidopsis thaliana seedlingsProc. Natl. Acad. Sci. USA8977917795PubMedGoogle Scholar
  11. Byrne, D. 1984Breeding cassavaPlant Breeding Rev.273134Google Scholar
  12. Calatayud, P.A., Le Ru, B. 1996Study of the nutritional relationships between cassava and mealybug and its host plantBull Soc. Zool. Fr. Evol. Zool.121391398Google Scholar
  13. Cliff, J., Lundquist, P., Mårtenssen, J., Rosling H. and Sörbo, B. 1985. Association of high cyanide and low sulphur intake in cassava-induced spastic paraperesis. Lancet ii: 1211–1213.Google Scholar
  14. Cock, J. 1985Cassava: New potential for a neglected cropWestfield PressLondonGoogle Scholar
  15. Conn, E. 1979Cyanogenic glycosidesInt. Rev. Biochem.272143Google Scholar
  16. Conn, E. 1994Cyanogenesis–a personal perspectiveActa Hort.3753143Google Scholar
  17. Delange, F., Ekpechi, L., Rosling, H. 1994Cassava cyanogenesis and iodine deficiency disorderActa Hort.375289293Google Scholar
  18. Dixon, A., Asiedu, R., Bokanga, M. 1994Breeding of cassava for low cyanogenic potential: problems, progress and prospectsActa Hort.375153161Google Scholar
  19. Du, L., Bokanga, M., Moller, B., Halkier, B. 1995The biosynthesis of cyanogenic glucosides in roots of cassavaPhytochem.39323326Google Scholar
  20. Elias, M., Sudhakaran, P., Nambisan, B. 1997aPurification and characterization of - cyanoalanine synthase from cassava tissuesPhytochem.46469472Google Scholar
  21. Elias, M., Nambisan, B., Sudhakaran, P. 1997bCatabolism of linamarin in cassavaPlant Sci.126155162Google Scholar
  22. Ernesto, M., Cardoso, A., Nicala, D., Mirione, E., Massaza, F., Cliff, J., Haque, M., Bradbury, J. 2002Persistent konzo and cyanogen toxicity from cassava in northern MozambiqueActa Trop.82357362PubMedGoogle Scholar
  23. Howlett, W., Brubaker, G., Mlingi, N., Rosling, H. 1990Konzo, an epidemic upper motor neuron disease studied in TanzaniaBrain113223235PubMedGoogle Scholar
  24. Hughes, M.A., Brown, K., Pancoro, A., Murray, B.S., Oxtoby, E., Hughes, J. 1992A molecular and biochemical analysis of the structure of the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Cranz)Arch. Biochem. Biophys.295273279PubMedGoogle Scholar
  25. Hughes, J., Carvahlo, F., Hughes, M. 1994Purification, characterization and cloning of ▮-hydroxynitrile lyase from cassava (Manihot esculenta Crantz)Arch. Biochem. Biophys.311496502PubMedGoogle Scholar
  26. Kawano, K., Narintaraporn, K., Narintaraporn, S., Sarakarn, S., Limsila, A., Watan-Anonta, W. 1998Yield improvement in a multistage breeding program for cassavaCrop Sci.38325332Google Scholar
  27. Kim, S., Gregory, D., Park, W. 1994Nuclear protein factors binding to a class-I patatin promoter region are tuber-specific and sucrose-induciblePlant Mol. Biol.26603615PubMedGoogle Scholar
  28. Koch, B., Nielsen, V., Halkier, B., Olsen, C., Møller, B. 1992The biosynthesis of cyanogenic glycosides in seedlings of cassava (Manihot esculenta Crantz)Arch. Biochem. Biophys.292141150PubMedGoogle Scholar
  29. P.J.Lea,R.D.Blackwell,K.W.Joy1992In: KMengel and D.H. Pillbeam (Eds.) Nitrogen Metabolism in Plants Claredon pressOxford, pp. 153–186Google Scholar
  30. P.J.Lea,S.A.Robinson,G.R.Stewart,1990In: (B.J Miflin and P.J. Lea(Eds.)) The Biochemistry of Plants vol. 16, Academic Press, San Diego. pp 121–159Google Scholar
  31. Lundquist, P., Rosling, H., Sörbo, B. 1985Determination of cyanide in whole blood, erythrocytes and plasmaClin. Chem.31591595PubMedGoogle Scholar
  32. Makame, M., Akoroda, M., Hahn, S. 1987Effects of reciprocal stem grafts on cyanide translocation in cassavaJ.␣Agr. Sci.109605608Google Scholar
  33. McMahon, J. 1997Physiological and biochemical analysis of factors regulating the synthesis of linamarin in the tropical plant cassava (Manihot esculenta Crantz), Ph.D thesisThe Ohio State University Columbus, OhioGoogle Scholar
  34. J. McMahon,R.Sayre,1994Regulation of cyanogenic potential in cassava (Manihot esculenta Crantz) In: W.M. Roca and A.M. Thro (Eds.) Proceedings of the Second International Scientific Meeting of the Cassava Biotechnology Networkpp. 423–438, Bogor, IndonesiaGoogle Scholar
  35. McMahon, J., White, W., Sayre, R. 1995Cyanogenesis in cassava (Manihot esculenta)J. Exp. Bot.46731741Google Scholar
  36. Mkpong, O., Yan, H., Chism, G., Sayre, R. 1990Purification, characterization, and localization of linamarase in cassavaPlant Physiol.93176181Google Scholar
  37. Mlingi, N , Kimatta, S., Rosling, H. 1991Konzo, a paralytic disease observed in southern TanzaniaTropical Doctor212425Google Scholar
  38. Nahrstedt, A. 1985Cyanogenic compounds as protecting agents for organismsPlant Syst. Evol.1503547Google Scholar
  39. Nartey, F. 1969Studies on cassava Manihot utillisima, biosynthesis of asparagines-14C from 14C-labelled hydrogen cyanide and its relations with cyanogenesisPhysiol. Plantarum.2210851096Google Scholar
  40. Nweke, F., Spencer, D., Lynam, J. 2002The Cassava transformation: Africa’s Best-Kept SecretMich. St. Univ. PressEast Lansing, USAGoogle Scholar
  41. Oluwole, O., Onabolu, A., Link, H., Roslin, H. 2000Persistence of tropical ataxic neuropathy in a Nigerian communityJ. Neurol. Neurosurg. Psych.6996101Google Scholar
  42. Osuntokun, B. 1981Cassava diet, chronic cyanide intoxification and neuropathy in Nigerian AfricansWorld Rev. Nutr. Diet.36141173PubMedGoogle Scholar
  43. Rosling, H. 1994Measuring effect in humans of dietary cyanide exposure from cassavaActa Hort.375271283Google Scholar
  44. Rosling, H., Mlingi, N., Tylleskar, T., Banea, M. 1993Causal mechanisms behind human diseases induced by cyanide exposure from cassavaRoca, W.M.Thro, A.M. eds. Proceedings of the first international scientific meeting of the Cassava Biotechnology NetworkCentro Internacional de Agricultura Tropical Cali, Colombia36637525–28 August 1992.Google Scholar
  45. Scott, G., Best, R., Rosegrant, M., Bokanga, M. 2002Roots and tubers in the global food system: a vision statement to the year 2020A co-publication of the International Potato CenterCentro Internacional de Agricultura Tropical, International Food Policy Research institute, International Institute of Tropical Agriculture and International Plant Genetic Resources Institute.Lima, PeruGoogle Scholar
  46. Selmar, D. 1994Translocation of cyanogenic glycosides in cassavaActa Hort.3756168Google Scholar
  47. Selmar, D., Lieberei, R., Biehl, R. 1988Mobilization and utilization of cyanogenic glycosides: the linustatin pathwayPlant Physiol.86711716Google Scholar
  48. Siritunga, D. 2002Generation of acyanogenic cassava (Manihot esculenta, Crantz): Transgenic approaches Ph. D. thesisThe Ohio State UniversityColumbus, OHGoogle Scholar
  49. Siritunga, D., Arias-Garcon, D., White, W., Sayre, R. 2004Over-expression of hydroxynitrile lyase in cassava roots accelerates cyanogenesis and detoxificationPlant Biotech. J.23743Google Scholar
  50. Siritunga, D., Sayre, R. 2003Generation of cyanogen-free transgenic cassavaPlanta217367373PubMedGoogle Scholar
  51. Sreeja, V., Nagahara, N., Li, Q., Minami, M. 2003New aspects in pathogenesis of konzo: neural cell damage directly caused by linamarin contained in cassava (Manihot esculenta Crantz)Brit. J. Nutr.90467472PubMedGoogle Scholar
  52. Tylleskar, T., Cooke, R., Banea, M., Poulter, N., Bikangi, N., Rosling, H. 1992Cassava cyanogens and konzo, an upper motor neuron disease found in AfricaLancet339208211PubMedGoogle Scholar
  53. White, W., Arias-Garzon, D., McMahon, J., Sayre, R.T. 1998Cyanogenesis in cassava: the role of hydroxynitrile lyase in root cyanide productionPlant Physiol.11612191225PubMedGoogle Scholar
  54. White, W., McMahon, J., Sayre, R. 1994Regulation of cyanogenesis in cassavaActa Hort.3756978Google Scholar
  55. Zourelidou, M., Torres-Zabala, M., Smith, C. and Bevan, M. 2002. Storekeeper defines a new class of plant-specific DNA-binding proteins.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  1. 1.The Ohio state universityColumbusUS

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