Transgenic Research

, Volume 13, Issue 5, pp 475–485 | Cite as

Expression of cold-tolerant pyruvate, orthophosphate dikinase cDNA, and heterotetramer formation in transgenic maize plants

  • Shozo Ohta
  • Yuji Ishida
  • Satoru Usami


Maize is a typical C4 plant of the NADP-malic enzyme type, and its high productivity is supported by the C4 photosynthetic cycle, which concentrates atmospheric CO2 in the leaves. The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced. Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C4 pathway in maize, loses its activity below about 12 °C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures. To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveriabrownii into maize by Agrobacterium-mediated transformation. We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F. bidentis and F. brownii with a maximum content of 1 mg/g fresh weight. In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK. Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved. Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 °C lower than that of the wild type.

antisense cold tolerance Flaveria brownii maize PPDK tetramer 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ashton AR, Burnell JN and Hatch MD (1984) Regulation of C4 photosynthesis: inactivation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and activation by phosphorolysis. Arch Biochem Biophys 230: 492-503.Google Scholar
  2. Ashton AR, Furbank RT and Trevanion SJ (1999) Antisense RNA inhibition of pyruvate, orthophosphate dikinase and NADP malate dehydrogenase in the C4 plant Flaveria bidentis: analysis of plants with a mosaic phenotype. Aust J Plant Physiol 26: 537-547.Google Scholar
  3. Bensen RJ, Johal GS, Crane VC, Tossberg JT, Schnable PS et al. (1995) Cloning and characterization of the maize an1 gene. Plant Cell 7: 75-84.Google Scholar
  4. Burnell JN (1990) A comparative study of the cold-sensitivity of pyruvate, Pi dikinase in Flaveria species. Plant Cell Physiol 31: 295-297.Google Scholar
  5. Burnell JN and Hatch MD (1985) Regulation of C4 photosynthesis: purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate, Pi dikinase. Arch Biochem Biophys 237: 490-503.Google Scholar
  6. Denecke J, Gosele J, Botterman J and Cornelissen M (1989) Quantitative analysis of transiently expressed gene in plant cells. Method Mol Cell Biol 1: 19-27.Google Scholar
  7. Du Y-C, Nose A and Wasano K (1999a) Effects of chilling temperature on photosynthetic rates, photosynthetic enzyme activities and metabolite levels in leaves of three sugarcane species. Plant Cell Environ 22: 317-324.Google Scholar
  8. Du Y-C, Nose A and Wasano K (1999b) Thermal characteristics of C4 photosynthetic enzymes from leaves of three sugarcane species differing in cold sensitivity. Plant Cell Physiol 40: 298-304.Google Scholar
  9. Fukayama H, Tsuchida H, Agarie S, Nomura M, Onodera H et al. (2001) Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiol 127: 1136-1146.Google Scholar
  10. Furbank RT, Chitty JA, Jenkins CLD, Taylor WC, Trevanion SJ et al. (1997) Genetic manipulation of key photosynthetic enzymes in the C4 plant Flaveria bidentis. Aust J Plant Physiol 24: 477-485.Google Scholar
  11. Getzoff TP, Zhu G, Bohnert HJ and Jensen RG (1998) Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation. Plant Physiol 116: 695-702.Google Scholar
  12. Glackin CA and Grula JW (1990) Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize. Proc Natl Acad Sci USA 87: 3004-3008.Google Scholar
  13. Hatch MD (1979) Regulation of C4 carbon pathway photosynthesis: factors affecting cold mediated inactivation and reactivation of pyruvate orthophosphate dikinase EC- Aust J Plant Physiol 6: 607-619.Google Scholar
  14. Hatch MD (1987) C4 photosynthesis: a unique blend of modi-fied biochemistry, anatomy and ultrastructure. Biochim Biophys Acta 895: 81-106.Google Scholar
  15. Hudspeth RL and Grula JW (1989) Structure and expression of the maize gene encoding the phosphoenolpyruvte carboxylase isozyme involved in C4 photosynthesis. Plant Mol Biol 12: 579-589.Google Scholar
  16. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T et al. (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14: 745-750.Google Scholar
  17. Jenkins CL and Hatch MD (1985) Properties and reaction mechanism of C4 leaf pyruvate, Pi dikinase. Arch Biochem Biophys 239: 53-62.Google Scholar
  18. Komari T, Hiei Y, Saito Y, Murai N and Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10: 165-174.Google Scholar
  19. Ku MS, Agarie S, Nomura M, Fukayama H, Tsuchida H et al. (1999) High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol 17: 76-80.Google Scholar
  20. Maruta Y, Ueki J, Saito H, Nitta N and Imaseki H (2002) Transgenic rice with reduced glutelin content by transformation with glutelin A antisense gene. Mol Breeding 8: 273-284.Google Scholar
  21. Matsuoka M (1990) Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize. J Biol Chem 265: 16772-16777.Google Scholar
  22. Matsuoka M and Numazawa T (1991) Cis-acting elements in the pyruvate, orthophosphate dikinase gene from maize. Mol Gen Genet 228: 143-152.Google Scholar
  23. Ohta S, Mita S, Hattori T and Nakamura K (1990) Construction and expression in tobacco of a b-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31: 805-813.Google Scholar
  24. Ohta S, Usami S, Ueki J, Kumashiro T, Komari T et al. (1996) Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate, orthophosphate dikinase. FEBS Lett 396: 152-156.Google Scholar
  25. Sheen J (1991) Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell 3: 225-245.Google Scholar
  26. Shirahashi K, Hayakawa S and Sugiyama T (1978) Cold lability of pyruvate, orthophosphate dikinase in the maize leaf. Plant Physiol 62: 826-830.Google Scholar
  27. Simon J-P (1996) Molecular forms and kinetic properties of pyruvate, Pi dikinase from two populations of barnyard grass (Echinochloa crus-galli) from sites of contrasting climates. Aust J Plant Physiol 23: 191-199.Google Scholar
  28. Sugiyama T (1973) Purification, molecular, and catalytic properties of pyruvate phosphate dikinase from the maize leaf. Biochemistry 12: 2862-2868.Google Scholar
  29. Sugiyama T and Boku K (1976) Differing sensitivity of pyruvate orthophosphate dikinase to low temperature in maize cultivars. Plant Cell Physiol 17: 851-854.Google Scholar
  30. Sugiyama T and Hirayama Y (1983) Correlation of activities of phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase with biomass in maize seedlings. Plant Cell Physiol 24: 783-787.Google Scholar
  31. Taniguchi M, Izawa K, Ku MS, Lin JH, Saito H et al. (2000a) The promoter for the maize C4 pyruvate, orthophosphate dikinase gene directs cell-and tissue-specific transcription in transgenic maize plants. Plant Cell Physiol 41: 42-48.Google Scholar
  32. Taniguchi M, Izawa K, Ku MS, Lin JH, Saito H et al. (2000b) Binding of cell type-specific nuclear proteins to the 50-flanking region of maize C4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells. Plant Mol Biol 44: 543-557.Google Scholar
  33. Terada R, Urawa H, Inagaki Y, Tsugane K and Iida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20: 1030-1034.Google Scholar
  34. Ueda K and Sugiyama T (1976) Purification and characterization of phosphoenolpyruvate carboxylase from maize leaves. Plant Physiol 57: 906-910.Google Scholar
  35. Ueki J, Ohta S, Morioka S, Komari T, Kuwata S et al. (1999) The synergistic effects of two-intron insertions on heterologous gene expression and advantages of the first intron of a rice gene for phospholipase D. Plant Cell Physiol 40: 618-623.Google Scholar
  36. Usami S, Ohta S, Komari T and Burnell JN (1995) Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii. Plant Mol Biol 27: 969-980.Google Scholar
  37. Usuda H (1984) Variations in the photosynthesis rate and activity of photosynthetic enzymes in maize leaf tissue of different ages. Plant Cell Physiol 25: 1297-1301.Google Scholar
  38. Usuda H, Ku MSB and Edwards GE (1984a) Activation of NADP-malate dehydrogenase, pyruvate, Pi dikinase, and fructose 1,6-bisphosphatase in relation to photosynthetic rate in maize. Plant Physiol 76: 238-243.Google Scholar
  39. Usuda H, Ku MSB and Edwards GE (1984b) Rates of photosynthesis relative to activity of photosynthetic enzymes chlorophyll and soluble protein content among ten 4-carbon pathway species. Aust J Plant Physiol 11: 509-517.Google Scholar
  40. Ward DA (1987) The temperature acclimation of photosynthetic response to CO2 in Zea mays and its relationship to the activities of photosynthetic enzymes and the CO2-concentrating mechanism of C4 photosynthesis. Plant Cell Environ 10: 407-411.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Shozo Ohta
    • 1
    • 2
    • 2
  • Yuji Ishida
    • 2
    • 3
  • Satoru Usami
    • 2
    • 3
  1. 1.API Company LimitedNagaragawa Research CenterNagaraJapan
  2. 2.Japan Tobacco Inc.Plant Breeding and Genetics Research LaboratoryShizuokaJapan
  3. 3.Japan Tobacco IncPlant Innovation CenterShizuokaJapan

Personalised recommendations