, Volume 120, Issue 1, pp 71–83 | Cite as

Isolation and characterisation of cDNAs encoding the large and small subunits of ADP-glucose pyrophosphorylase from cassava (Manihot esculenta Crantz)

  • Tichafa R.I. Munyikwa
  • Jan Kreuze
  • Martin Fregene
  • Luc Suurs
  • Evert Jacobsen
  • Richard G.F. Visser


Screening of a tuber specific cassava cDNA library resulted in the isolation of full length cDNA clones with homology to the genes encoding the small and large subunits of ADP glucose pyrophosphoryalse. Sequence analysis revealed that AGPase B the clone with homology to the small subunit shared 54% homology at amino acid level with the AGPase S clone that is more closely related to the large subunit. Segregation analysis of a cross between the cassava cultivars TMS 30572 and CM 2177-2 revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. AGPase B is a low copy gene of which one member is localised on the female derived linkage group P. The two genes are expressed in all cassava tissues but AGPase B exhibits a higher steady state mRNA level than AGPase S and is highly expressed in leaf and tuber tissue. The AGPase enzyme activity was much higher in young cassava leaves as compared to older leaves and tubers. Cassava AGPase was activated by 3-PGA and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA but was highly inhibited by Pi. Transformation of potato (Solanum tuberosum) plants with an antisense AGPase B construct resulted in 10 out of 134 antisense AGPase B plants having on average 3.5 times more tubers than the control non transgenic plants. Analysis of these transgenic plants revealed they had greatly reduced levels of AGPase B mRNA, 1.5 to 3 times less starch, and five times higher levels of soluble sugars, sucrose, glucose and fructose, to those found in control plants.

AGPase B AGPase S ADP-glucose pyrophosphorylase antisense cassava potato 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Altschul, S.F., W. Gish, W. Miller, E.W. Myers & D.J. Lipman, 1990. Basic local alignment search tool. J Mol Biol 215: 403–410.PubMedCrossRefGoogle Scholar
  2. Bae, J.M., M. Giroux & L.C. Hannah, 1990. Cloning and characterisation of the brittle-2 gene of maize. Maydica 35: 317–322.Google Scholar
  3. Bae, J.M. & J.R. Liu, 1997. Molecular cloning and characterisation of two novel isoforms of the small subunit of ADPglucose pyrophosphorylase from sweet potato. Mol Gen Genet 254: 179–185.PubMedCrossRefGoogle Scholar
  4. Bhave, M.R., S. Lawrence, C. Barton & L.C. Hannah, 1990. Identification and molecular characterisation of shrunken-2 cDNA clones of maize. Plant Cell 2: 581–588.PubMedCrossRefGoogle Scholar
  5. Copeland, L. & J. Preiss, 1991. Purification of spinach leaf ADPglucose pyrophosphorylase. Plant Physiol 68: 996–1001.CrossRefGoogle Scholar
  6. Feinberg, A.P. & B. Volgestein, 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13.PubMedCrossRefGoogle Scholar
  7. Fregene, M., F. Angel, R. Gomez, F. Rodriguez, P. Chavariaga, W. Roca, J. Tohme & M. Bonierbale, 1997. A molecular genetic map of cassava (Manihot esculenta Crantz. Theor Appl Genet 95: 431–441.CrossRefGoogle Scholar
  8. Guerineau, F. & P.M. Mullineaux, 1993. Plant transformation and expression vectors. In: R.R.D. Croy (Ed.), Plant Molecular Biology Labfax, pp. 121–148, BIOS Scientific, Oxford.Google Scholar
  9. Hannah, L.C., D.M. Tuschall & R.J. Mans, 1980. Multiple forms of maize endosperm ADP-glucose pyrophosphorylase and their control by shrunken-2 and brittle-2. Genetics 95: 961–970.PubMedGoogle Scholar
  10. Iglesias, A.A, G.F. Barry, C. Meyer, L. Blocksberg, P.A. Nakata, T. Green, M.J. Laughlin, T.W. Okita, G.M. Kishore & J. Preiss, 1993. Expression of the potato tuber ADPglucose pyrophosphorylase in Escherichia coli. J Biol Chem 269: 1081–1086.Google Scholar
  11. Kleczkowski, L.A., P. Villand, U. Lüthi, O.A. Olsen & J. Preiss, 1993. Insensitivity of barley endosperm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation. Plant Physiol 101: 179–186.PubMedCrossRefGoogle Scholar
  12. Kuipers, A. 1994. Antisense RNA Mediated Inhibition of Granule Bound Starch Synthase Expression in Potato. PhD Thesis Wageningen Agricultural University, pp. 125.Google Scholar
  13. Kuipers, A., J.J. Soppe, E. Jacobsen & R.G.F. Visser, 1995. Factors affecting the inhibition of granule bound starch synthase gene expression in potato via antisense RNA. Mol Gen Genet 246: 745–755.PubMedCrossRefGoogle Scholar
  14. La Cognita, U., L. Willmitzer & B. Müller-Röber, 1995. Molecular cloning and characterization of novel isoforms of potato ADPglucose pyrophosphorylase. Mol Gen Genet 246: 538–548.CrossRefGoogle Scholar
  15. Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincols & L. Newburg, 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174–181.PubMedCrossRefGoogle Scholar
  16. Lin, T.P., T. Caspar, C. Somerville & J. Preiss, 1988. A starch deficient mutant of Arabidopsis thaliana with low ADP-glucose pyrophosphorylase activity lacks one of the two subunits of the enzyme. Plant Physiol 88: 1175–1181.PubMedGoogle Scholar
  17. Lloyd, J., F. Springer, A. Buleon, B. Müller-Röber, L. Willmitzer & J. Kossmann, 1999. The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers. Planta 209: 230–238.PubMedCrossRefGoogle Scholar
  18. Luo, C. & L.A. Kleczkowski, 1999. Expression of barley ADPglucose pyrophosphorylase in Escherichia coli: processing and regulatory considerations. Phytochemistry 50: 209–214.PubMedCrossRefGoogle Scholar
  19. Müller-Röber, B.T., J. Kossmann, L.C. Hannah, L. Willmitzer & U. Sonnewald, 1990. One of two different ADP-glucose pyrophosphorylase genes responds strongly to elevated levels of sucrose. Mol Gen Genet 224: 136–146.PubMedCrossRefGoogle Scholar
  20. Müller-Röber, B.T., U. Sonnewald & L. Willmitzer, 1992. Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J 11: 1229–1238.PubMedGoogle Scholar
  21. Munyikwa, T.R.I., C.J.J.M. Raemakers, M. Schreuder, R. Kok, M. Schippers, E. Jacobsen & R.G.F. Visser, 1998. Pinpointing towards improved transformation and regeneration of cassava (Manihot esculenta Crantz. Plant Science 135: 87–101.CrossRefGoogle Scholar
  22. Nakamura, Y., Kawaguchi, K. 1992. Multiple forms of ADPglucose pyrophosphorylase of rice endosperm. Physiol Plant 84: 336–342.CrossRefGoogle Scholar
  23. Nakata, P.A., T.W. Greene, J.M. Anderson, B.J. Smith-White, T.W. Okita & J. Preiss, 1991. Comparison of the primary structure of two potato tuber ADP-glucose pyrophosphorylase subunits. Plant Mol Biol 17: 1089–1093.PubMedCrossRefGoogle Scholar
  24. Okita, T.W., P.A. Nakata, J.M. Anderson, J. Sowokinos, M. Morell & J. Preiss, 1990. The subunit structure of potato ADP-glucose pyrophosphorylase. Plant Physiol 93: 785–790.PubMedGoogle Scholar
  25. Preiss, J., K. Ball, J. Hutney, B. Smith-White, L. Li, & T.W. Okita, 1991. Regulatory mechanisms involved in the biosynthesis of starch. Pure Appl Chem 63: 535–544.Google Scholar
  26. Prioul, J.L., E. Jeanette A. Reyss, N. Gregory, M. Giroux, L.C. Hannah & M. Causse, 1994. Expression of ADPglucose pyrophosphorylase in maize (Zea mays L.) grain and leaf source during grain filling. Plant Physiol 104: 179–187.PubMedCrossRefGoogle Scholar
  27. Röber, M., Geider, K., Müller-Röber B. & L. Willmitzer 1996. Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates. Planta 199: 528–536.PubMedCrossRefGoogle Scholar
  28. Salehuzzaman, S.N.I.M., E., Jacobsen & R.G.F. Visser, 1993. Isolation and characterization of cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato. Plant Mol Biol 23: 947–962.PubMedCrossRefGoogle Scholar
  29. Salehuzzaman, S.N.I.M., E. Jacobsen & R.G.F. Visser, 1992. Cloning, partial sequencing and expression of a cDNA coding for branching enzyme in cassava (Manihot esculenta Crantz). Plant Mol Biol 20: 809–819.PubMedCrossRefGoogle Scholar
  30. Sambrook, J., T. Maniatis & E.F. Fritsch, 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour, New York, Cold Spring Harbour Laboratory.Google Scholar
  31. Sanger, F., S. Nicklen & A.R. Coulson, 1977. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467.PubMedCrossRefGoogle Scholar
  32. Smith-White, B.J. & J. Preiss, 1992. Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources. J Mol Evol 34: 449–464.PubMedCrossRefGoogle Scholar
  33. Villand, P., O.A. Olsen & L.A. Kleczkowski, 1993. Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana. Plant Mol Biol 23: 1279–1284.PubMedCrossRefGoogle Scholar
  34. Visser, R.G.F., 1991. Regeneration and Transformation by Agrobacterium tumefaciens. Plant Tissue Culture Manual B5: 1–9. Kluwer Academic Publishers.Google Scholar
  35. Visser, R.G.F., L.C.M. Suurs, P.A.M. Steeneken & E. Jacobsen, 1997. Some physicochemical properties of amylose-free potato starch. Starch/Staerke 49: 443–448.Google Scholar
  36. Weber, H., U. Heim, L. Borisjuk & U. Wobus, 1995. Cell-type, specific coordinate expression of two ADPglucose pyrophosphorylase genes in relation to starch biosynthesis during seed development in Vicia faba L. Planta195: 352–361.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Tichafa R.I. Munyikwa
    • 1
  • Jan Kreuze
    • 1
  • Martin Fregene
    • 2
  • Luc Suurs
    • 1
  • Evert Jacobsen
    • 1
  • Richard G.F. Visser
    • 1
  1. 1.Laboratory of Plant Breeding, Graduate School Experimental Plant SciencesWageningen UniversityWageningenThe Netherlands
  2. 2.Biotechnology UnitCIATCaliColombia

Personalised recommendations