Skip to main content
SpringerLink
Log in

High-Level Expression of Cold-Tolerant Pyruvate, Orthophosphate Dikinase from a Genomic Clone with Site-Directed Mutations in Transgenic Maize

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C4 photosynthesis of maize under cold conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

PEPC:

Phosphoenolpyruvate carboxylase

PPDK:

Pyruvate, orthophosphate dikinase

References

  • Aoyagi K, Bassham JA, Greene FC (1984) Pyruvate orthophosphate dikinase gene expression in developing wheat seeds. Plant Physiol 75:393–396

    PubMed  CAS  Google Scholar 

  • Ashton AR, Burnell JN, 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

    Article  PubMed  CAS  Google Scholar 

  • Burnell JN (1990) A comparative study of the cold-sensitivity of pyruvate, Pi dikinase in Flaveria species. Plant Cell Physiol 31:295–297

    CAS  Google Scholar 

  • Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1:1183–1200

    Article  PubMed  CAS  Google Scholar 

  • Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689

    Article  PubMed  CAS  Google Scholar 

  • Denecke J, Gosele J, Botterman J, Cornelissen M (1989) Quantitative analysis of transiently expressed gene in plant cells. Methods Mol Cell Biol 1:19–27

    CAS  Google Scholar 

  • Du Y-C, Nose A, Wasano K (1999) Thermal characteristics of C4 photosynthetic enzymes from leaves of three sugarcane species differing in cold sensitivity. Plant Cell Physiol 40:298–304

    CAS  Google Scholar 

  • Fukayama H, Tsuchida H, Agarie S, Nomura M, Onodera H, Ono K, Lee BH, Hirose S, Toki S, Ku MS, Makino A, Matsuoka M, Miyao M (2001) Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiol 127:1136–1146

    Article  PubMed  CAS  Google Scholar 

  • Glackin CA, 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

    Article  PubMed  CAS  Google Scholar 

  • Hatch MD (1979) Regulation of C4 carbon pathway photosynthesis: factors affecting cold mediated inactivation and reactivation of pyruvate orthophosphate dikinase EC-2.7.9.1. Aust J Plant Physiol 6:607–619

    CAS  Google Scholar 

  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 15:51–59

    Article  Google Scholar 

  • Imaizumi N, Ku MS, Ishihara K, Samejima M, Kaneko S, Matsuoka M (1997). Characterization of the gene for pyruvate, orthophosphate dikinase from rice, a C3 plant, and a comparison of structure and expression between C3 and C4 genes for this protein. Plant Mol Biol 34:701–716

    Article  PubMed  CAS  Google Scholar 

  • Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750

    Article  PubMed  CAS  Google Scholar 

  • Ishida Y, Murai N, Kuraya Y, Ohta S, Saito H, Hiei Y, Komari T (2004) Improved co-transformation of maize with vectors carrying two separate T-DNAs mediated by Agrobacterium tumefaciens. Plant Biotechnol 21:57–63

    CAS  Google Scholar 

  • Jenkins CL, Hatch MD (1985) Properties and reaction mechanism of C4 leaf pyruvate, Pi dikinase. Arch Biochem Biophys 239:53–62

    Article  PubMed  CAS  Google Scholar 

  • Kawabe A, Miyashita NT (2003) Patterns of codon usage bias in three dicot and four monocot plant species. Genes Genet Syst 78:343–352

    Article  PubMed  CAS  Google Scholar 

  • Komari T, Hiei Y, Saito Y, Murai N, 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

    Article  PubMed  CAS  Google Scholar 

  • Ku MS, Kano-Murakami Y, Matsuoka M (1996) Evolution and expression of C4 photosynthesis genes. Plant Physiol 111:949–957

    Article  PubMed  CAS  Google Scholar 

  • Ku MS, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M (1999) High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol 17:76–80

    Article  PubMed  CAS  Google Scholar 

  • Matsuoka M (1990) Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize. J Biol Chem 265:16772–16777

    PubMed  CAS  Google Scholar 

  • Matsuoka M (1995) The gene for pyruvate, orthophosphate dikinase in C4 plants: structure, regulation and evolution. Plant Cell Physiol 36:1–7

    Google Scholar 

  • McElroy D, Zhang W, Cao J, Wu R (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2:163–171

    Article  PubMed  CAS  Google Scholar 

  • Murray EE, Lotzer J, Eberle M (1989) Codon usage in plant genes. Nucleic Acids Res 17:477–498

    Article  PubMed  CAS  Google Scholar 

  • Naidu SL, Moose SP, Al-Shoaibi AK, Raines CA, Long SP (2003) Cold tolerance of C4 photosynthesis in Miscanthus × giganteus: adaptation in amounts and sequence of C4 photosynthetic enzymes. Plant Physiol 132:1688–1697

    Article  PubMed  CAS  Google Scholar 

  • Ohta S, Usami S, Ueki J, Kumashiro T, Komari T, Burnell JN (1996) Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate, orthophosphate dikinase. FEBS Lett 396:152–156

    Article  PubMed  CAS  Google Scholar 

  • Ohta S, Ishida Y, Usami S (2004) Expression of cold-tolerant pyruvate, orthophosphate dikinase cDNA, and heterotetramer formation in transgenic maize plants. Transgenic Res 13:475–485

    Article  PubMed  CAS  Google Scholar 

  • Rosche E, Westhoff P (1995) Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C4 plant Flaveria trinervia (Asteraceae). Plant Mol Biol 29:663–678

    Article  PubMed  CAS  Google Scholar 

  • Sheen J (1991) Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell 3:225–245

    Article  PubMed  CAS  Google Scholar 

  • Shirahashi K, Hayakawa S, Sugiyama T (1978) Cold lability of pyruvate,orthophosphate dikinase in the maize leaf. Plant Physiol 62:826–830

    PubMed  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • Sugiyama T (1973) Purification, molecular, and catalytic properties of pyruvate phosphate dikinase from the maize leaf. Biochemistry 12:2862–2868

    Article  PubMed  CAS  Google Scholar 

  • Sugiyama T, Boku K (1976) Differing sensitivity of pyruvate orthophosphate dikinase to low temperature in maize cultivars. Plant Cell Physiol 17:851–854

    CAS  Google Scholar 

  • Sugiyama T, Hirayama Y (1983) Correlation of activities of phosphoenolpyruvate carboxylase and pyruvate,orthophosphate dikinase with biomass in maize seedlings. Plant Cell Physiol 24:783–787

    CAS  Google Scholar 

  • Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K (1990) Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron. Nucleic Acids Res 18:6767–6770

    Article  PubMed  CAS  Google Scholar 

  • Usami S, Ohta S, Komari T, Burnell JN (1995) Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii. Plant Mol Biol 27:969–980

    Article  PubMed  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • Usuda H, Ku MSB, 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

    Article  CAS  Google Scholar 

  • Usuda H, Ku MSB, 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

    Article  CAS  Google Scholar 

  • Vasil V, Clancy M, Ferl RJ, Vasil KI, Hannah LC (1989) Increased gene expression by the first intron of maize shrunken-1 locus in grass species. Plant Physiol 91:1575–1579

    PubMed  CAS  Google Scholar 

  • Wang M-B, Waterhouse PM (1997) A rapid and simple method of assaying plants transformed with hygromycin and PPT resistance genes. Plant Mol Biol Rep 15:209–215

    Article  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. M. Matsuoka for kindly providing the genomic clone of maize PPDK. We also thank Dr. T. Komari for critical reading of the manuscript, and Ms. M. Tabayashi-Kuramoto, Ms. A.␣Yamashita, and Ms E. Usami for their technical assistance.

Author information

Authors and Affiliations

  1. Plant Breeding and Genetics Research Laboratory, Japan Tobacco, 700 Higashibara, Toyoda, Iwata, Shizuoka, 438-0802, Japan

    Shozo Ohta, Yuji Ishida & Satoru Usami

  2. Nagaragawa Research Center, API Company, 692-3 Yamasaki, Nagara, Gifu, 502-0071, Japan

    Shozo Ohta

  3. Plant Innovation Center, Japan Tobacco, 700 Higashibara, Toyoda, Iwata, Shizuoka, 438-0802, Japan

    Yuji Ishida & Satoru Usami

Authors
  1. Shozo Ohta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuji Ishida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satoru Usami
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shozo Ohta.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ohta, S., Ishida, Y. & Usami, S. High-Level Expression of Cold-Tolerant Pyruvate, Orthophosphate Dikinase from a Genomic Clone with Site-Directed Mutations in Transgenic Maize. Mol Breeding 18, 29–38 (2006). https://doi.org/10.1007/s11032-006-9011-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-006-9011-8

Keywords

Search

Navigation