Abstract
Clones encoding two different forms of plastid pyruvate kinase (PKp; EC 2.7.1.40) have been isolated from both castor and tobacco seed cDNA libraries. One form, designated PKpA, from castor was described in a previous report, and the tobacco homologue of PKpA has now been isolated. In addition, a second cDNA, designated PKpG, has been identified and sequenced in both species. Western blot analysis, using antibodies raised against protein overexpressed from these clones, indicates that they encode the two predominant polypeptides of plastid pyruvate kinase from developing castor endosperm. In castor, both PKpA and PKpG are encoded by single genes. In the allotetraploid Nicotiana tabacum, there are two copies of each, one derived from each of the progenitors of this species. The expression of the genes for PKpA and PKpG was examined in various tissues from both castor and tobacco. In castor, both forms are expressed in developing and germinating endosperm and in the root but neither is expressed in the leaf. In tobacco, both forms are expressed in developing seeds but in mature tissues, PKpA is most abundant in roots and PKpG in leaves.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bennett J: Protein phosphorylation in green plant chloroplasts. Ann Rev Plant Physiol 42: 281–311 (1991).
Blakeley SD, Dennis DT: Molecular approaches to the manipulation of carbon allocation in plants. Can J Bot 71: 765–778 (1993).
Blakelley SD, Plaxton WC, Dennis DT: Cloning and characterisation of a cDNA for the cytosolic isozyme of plant pyruvate kinase: the relationship between the plant and non-plant enzyme. Plant Mol Biol 15: 665–669 (1990).
Blakeley SD, Plaxton WC, Dennis DT: Relationship between the subunits of leucoplast pyruvate kinase from Ricinus communis and a comparison with the enzyme from other sources. Plant Physiol 96: 1283–1288 (1991).
Boyle SA, Hemmingsen SM, Dennis DT: Uptake and processing of the precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase by leucoplasts from the endosperm of developing castor oil seeds. Plant Physiol 81: 817–822 (1986).
Chua N-H, Bartlett SG, Weiss M: Preparation and characterisation of antibodies to chloroplast proteins. In: Edelman M, Hallick RB, Chua N-H (eds) Methods in Chloroplast Molecular Biology, pp. 1063–1080. Elsevier Biomedical Press, Amsterdam (1982).
Cognet M, Bergot M-O, Kahn A: Cis-acting DNA elements regulating expression of the liver pyruvate kinase gene in hepatocytes and hepatoma cells. Evidence for tissue specific activators and extinguisher. J Biol Chem 266: 7368–7375 (1991).
Cole KP, Blakeley SD, Dennis DT: Structure of the gene encoding potato cytosolic pyruvate kinase. Gene 122: 255–261 (1992).
Dennis DT, Blakeley SD: Carbon partitioning in developing oilseeds. In: Mackenzie SL, Taylor DC (eds) Seed oils for the future. A.O.C.S. Press, Champaign, IL (1993).
Dennis DT, Miernyk JA: Compartmentation of non-photosynthetic carbohydrate metabolism. Ann Rev Plant Physiol 33: 27–50 (1982).
Greenwood JS, Bewley JD: Seed development in Ricinus communis (castor bean), I. Descriptive morphology. Can J Bot 60: 1751–1760 (1982).
Hattori J, Gottlob-McHugh SG, Johnson DA: Isolation of high molecular weight DNA from plants. Anal Biochem 165: 70–74 (1987).
Huynh TV, Young RA, Davis RW: Constructing and screening cDNA libraries in λgt10 and λgt11. In: Glover DM (ed) DNA Cloning: A Practical Approach, vol. 1, pp. 49–78, IRL Press, Oxford (1988).
Imarai M, Hinrichsen P, Bazaes S, Wilkens M, Eyzaguirre J: Yeast pyruvate kinase: essential lysine residues in the active site. Int J Biochem 20: 1001–1008 (1988).
Inoue H, Noguchi T, Tanaka T: Complete aa sequence of rat L-type pyruvate kinase deduced from the cDNA sequence. Eur J Biochem 154: 465–469 (1986).
Ireland RJ, DeLuca V, Dennis DT: Isoenzymes of pyruvate kinase in etioplasts and chloroplasts. Plant Physiol 63: 903–907 (1979).
Johnson SC, Bailey T, Becker RR, Cardenas JM: Isolation and sequence determination of a peptide located at or near the active site of bovine muscle pyruvate kinase. Biochem Biophys Res Commun 90: 525–530 (1979).
Laemmli UK: Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lehrach HD, Diamond D, Wozney JM, Boedtker H: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reevaluation. Biochemistry 16: 4743–4751 (1977).
Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16–20 (1987).
Lone Y-C, Simon M-P, Kahn A, Marie J: Complete nucleotide and deduced aa sequences of rat L-type pyruvate kinase. FEBS Lett 195: 97–100 (1986).
MacDonald RJ, Switt GH, Przybyla AE, Chirgwin JM: Isolation of DNA using guanidinium salts. Meth Enzymol 152: 219–227 (1987).
Marie J, Simon M-P, Lone Y-C, Cognet M, Kahn A: Tissue-specific heterogeneity of the 3′-untranslated region of L-type pyruvate kinase mRNA's. Eur J Biochem 158: 33–41 (1986).
Muirhead H, Clayden DA, Cuffe CP, Davies C: Crystallographic studies on the structure and catalytic activity of pyruvate kinase from skeletal muscle. Biochem Soc Trans 15: 996–999 (1987).
Munnich A, Lyonnet S, Chauvet D, Van Schaftingen E, Kahn A: Differential effects of glucose and fructose on liver L-type pyruvate kinase gene expression in vivo. J Biol Chem 262: 17065–17071 (1987).
Noguchi T, Inoue H, Tanaka T: The M1- and M2-isozymes of rat pyruvate kinase are produced from the same gene by alternative splicing. J Biol Chem 261: 13807–13812 (1986).
Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T: The L- and R-isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J Biol Chem 262: 14366–14371 (1987).
Noguchi T, Yamada K, Yamagata K, Takenaka M, Nakajima H, Imai E, Wang Z, Tanaka T: Expression of liver type pyruvate kinase in insulinoma cells: involvement of LF-B1 (HNF1). Biochem Biophys Res Comm 181: 259–264 (1991).
Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448 (1988).
Plaxton WC: Purification of pyruvate kinase from germinating castor bean endosperm. Plant Physiol 86: 1064–1069 (1988).
Plaxton WC: Molecular and immunological characterisation of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf. Eur J Biochem 181: 443–451 (1989).
Plaxton WC: Leucoplast pyruvate kinase from developing castor oil seeds. Characterisation of the enzyme's degradation by a cysteine endopeptidase. Plant Physiol 97: 1334–1338 (1991).
Plaxton WC, Dennis DT, Knowles VK: Purification of leucoplast pyruvate kinase from developing castor bean endosperm. Plant Physiol 94: 1528–1534 (1990).
Podesta FE, Plaxton WC: Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds. Eur J Biochem 279: 495–501 (1991).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY (1989).
Sangwan RS, Gauthier DA, Turpin DH, Pomeroy MK, Plaxton WC: Pyruvate kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napu. Planta 187: 198–202 (1992).
Smith RG, Gauthier DA, Dennis DT, Turpin DH: Malate- and pyruvate-dependent fatty acid synthesis in leucoplasts from developing castor endosperm. Plant Physiol 98: 1233–1238 (1992).
Vaulont S, Munnich A, Decaux J-F, Kahn A: Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver. J Biol Chem 261: 7621–7625 (1986).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Blakeley, S., Gottlob-McHugh, S., Wan, J. et al. Molecular characterization of plastid pyruvate kinase from castor and tobacco. Plant Mol Biol 27, 79–89 (1995). https://doi.org/10.1007/BF00019180
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00019180