Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1992 Oct;100(2):820–825. doi: 10.1104/pp.100.2.820

Normal Growth of Transgenic Tobacco Plants in the Absence of Cytosolic Pyruvate Kinase 1

Sylvia G Gottlob-McHugh 1,2, Rajender S Sangwan 1,2,2, Stephen D Blakeley 1,2, Greg C Vanlerberghe 1,2,3, Kenton Ko 1,2, David H Turpin 1,2,4, William C Plaxton 1,2, Brian L Miki 1,2, David T Dennis 1,2
PMCID: PMC1075631  PMID: 16653063

Abstract

The coding sequence of the cytosolic isozyme of potato tuber pyruvate kinase (PK) was attached to the transit peptide of the small subunit of pea ribulose-1,5-bisphosphate carboxylase oxygenase and placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Nicotiana tabacum. Unexpectedly, two primary transformants were recovered in which PK activity in leaves was greatly reduced. The reduction in PK activity appeared to result from the complete absence of the cytosolic form of the enzyme (PKc). In addition, no PKc could be detected on western blots of leaf extracts. Metabolite analyses indicated that the levels of phosphoenolpyruvate are substantially higher in PKc-deficient leaves than in wild-type leaves, consistent with a block in glycolysis at the step catalyzed by PK. PKc deficiency in the leaves does not appear to adversely affect plant growth. Analysis of progeny indicates that PKc deficiency is a heritable trait. The leaves of PKc-deficient transformants have normal rates of photosynthetic O2 evolution and respiratory O2 consumption, indicating that these plants are using alternative pathways to bypass PK.

Full text

PDF
820

Images in this article

822Figure 2
on p.822
823Figure 4
on p.823

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
  2. Blakeley S. D., Plaxton W. C., Dennis D. T. Cloning and characterization of a cDNA for the cytosolic isozyme of plant pyruvate kinase: the relationship between the plant and non-plant enzyme. Plant Mol Biol. 1990 Oct;15(4):665–669. doi: 10.1007/BF00017842. [DOI] [PubMed] [Google Scholar]
  3. Blakeley S. D., Plaxton W. C., Dennis D. T. Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources. Plant Physiol. 1991 Aug;96(4):1283–1288. doi: 10.1104/pp.96.4.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Duff S. M., Lefebvre D. D., Plaxton W. C. Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells. Plant Physiol. 1989 Jun;90(2):734–741. doi: 10.1104/pp.90.2.734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duff S. M., Moorhead G. B., Lefebvre D. D., Plaxton W. C. Phosphate Starvation Inducible ;Bypasses' of Adenylate and Phosphate Dependent Glycolytic Enzymes in Brassica nigra Suspension Cells. Plant Physiol. 1989 Aug;90(4):1275–1278. doi: 10.1104/pp.90.4.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  7. Gamborg O. L., Miller R. A., Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968 Apr;50(1):151–158. doi: 10.1016/0014-4827(68)90403-5. [DOI] [PubMed] [Google Scholar]
  8. Goring D. R., Thomson L., Rothstein S. J. Transformation of a partial nopaline synthase gene into tobacco suppresses the expression of a resident wild-type gene. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1770–1774. doi: 10.1073/pnas.88.5.1770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hattori J., Gottlob-McHugh S. G., Johnson D. A. The isolation of high-molecular-weight DNA from plants. Anal Biochem. 1987 Aug 15;165(1):70–74. doi: 10.1016/0003-2697(87)90202-8. [DOI] [PubMed] [Google Scholar]
  10. Ireland R. J., Deluca V., Dennis D. T. Isoenzymes of pyruvate kinase in etioplasts and chloroplasts. Plant Physiol. 1979 May;63(5):903–907. doi: 10.1104/pp.63.5.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jefferson R. A., Kavanagh T. A., Bevan M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987 Dec 20;6(13):3901–3907. doi: 10.1002/j.1460-2075.1987.tb02730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  13. Lin M., Turpin D. H., Plaxton W. C. Pyruvate kinase isozymes from the green alga, Selenastrum minutum. II. Kinetic and regulatory properties. Arch Biochem Biophys. 1989 Feb 15;269(1):228–238. doi: 10.1016/0003-9861(89)90104-5. [DOI] [PubMed] [Google Scholar]
  14. MacDonald R. J., Swift G. H., Przybyla A. E., Chirgwin J. M. Isolation of RNA using guanidinium salts. Methods Enzymol. 1987;152:219–227. doi: 10.1016/0076-6879(87)52023-7. [DOI] [PubMed] [Google Scholar]
  15. Maitra P. K., Lobo Z. Pyruvate kinase mutants of Saccharomyces cerevisiae: biochemical and genetic characterisation. Mol Gen Genet. 1977 Apr 29;152(3):193–200. doi: 10.1007/BF00268817. [DOI] [PubMed] [Google Scholar]
  16. Miwa S. Pyruvate kinase deficiency. Prog Clin Biol Res. 1990;344:843–852. [PubMed] [Google Scholar]
  17. Napoli C., Lemieux C., Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990 Apr;2(4):279–289. doi: 10.1105/tpc.2.4.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pertierra A. G., Cooper R. A. Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants. J Bacteriol. 1977 Mar;129(3):1208–1214. doi: 10.1128/jb.129.3.1208-1214.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Plaxton W. C., Dennis D. T., Knowles V. L. Purification of leucoplast pyruvate kinase from developing castor bean endosperm. Plant Physiol. 1990 Dec;94(4):1528–1534. doi: 10.1104/pp.94.4.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Plaxton W. C. Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf. Eur J Biochem. 1989 May 1;181(2):443–451. doi: 10.1111/j.1432-1033.1989.tb14745.x. [DOI] [PubMed] [Google Scholar]
  21. Plaxton W. C. Purification of pyruvate kinase from germinating castor bean endosperm. Plant Physiol. 1988 Apr;86(4):1064–1069. doi: 10.1104/pp.86.4.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Podestá F. E., Plaxton W. C. Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds. Biochem J. 1991 Oct 15;279(Pt 2):495–501. doi: 10.1042/bj2790495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith C. J., Watson C. F., Bird C. R., Ray J., Schuch W., Grierson D. Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet. 1990 Dec;224(3):477–481. doi: 10.1007/BF00262443. [DOI] [PubMed] [Google Scholar]
  24. Theodorou M. E., Elrifi I. R., Turpin D. H., Plaxton W. C. Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum. Plant Physiol. 1991 Apr;95(4):1089–1095. doi: 10.1104/pp.95.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vanlerberghe G. C., Feil R., Turpin D. H. Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product. Plant Physiol. 1990 Nov;94(3):1116–1123. doi: 10.1104/pp.94.3.1116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van der Krol A. R., Mur L. A., Beld M., Mol J. N., Stuitje A. R. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell. 1990 Apr;2(4):291–299. doi: 10.1105/tpc.2.4.291. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES