Planta

, Volume 223, Issue 5, pp 901–909 | Cite as

Arabidopsis phosphoenolpyruvate carboxylase genes encode immunologically unrelated polypeptides and are differentially expressed in response to drought and salt stress

  • Rosario Sánchez
  • Amando Flores
  • Francisco Javier Cejudo
Original Article

Abstract

The phosphoenolpyruvate carboxylase (PEPC) gene family of Arabidopsis is composed of four genes. Based on sequence analysis it was deduced that Atppc1, Atppc2 and Atppc3 genes encode plant-type PEPCs, whereas Atppc4 encodes a PEPC without phosphorylation motif, but no data at the protein level have been reported. Here, we describe the analysis of the four Arabidopsis PEPC polypeptides, which were expressed in Escherichia coli. Immunological characterization with anti plant-type PEPC and an anti-AtPPC4 antibody, raised in this work, showed that the bacterial-type PEPC is unrelated with plant-type PEPCs. Western-blot analysis of different Arabidopsis organs probed with anti plant-type PEPC antibodies detected a double band, the one with low molecular weight corresponding to the three plant-type PEPCs. The high molecular weight subunit is not encoded by any of the Arabidopsis PEPC genes. No bands were detected with the anti-AtPPC4 antibody. PEPC genes show differential expression in Arabidopsis organs and in response to environmental stress. Atppc2 transcripts were found in all Arabidopsis organs suggesting that it is a housekeeping gene. In contrast, Atppc3 gene was expressed in roots and Atppc1 in roots and flowers, as Atppc4. Highest PEPC activity was found in roots, which showed expression of the four PEPC genes. Salt and drought exerted a differential induction of PEPC gene expression in roots, Atppc4 showing the highest induction in response to both stresses. These results show that PEPC is part of the adaptation of the plant to salt and drought and suggest that this is the function of the new bacterial-type PEPC.

Keywords

Abiotic stress Arabidopsis Gene family Phosphoenolpyruvate carboxylase Root Rosette leaves 

Abbreviations

IPTG:

Isopropyl β-d-thiogalactoside

PEPC:

Phosphoenolpyruvate carboxylase

References

  1. Agetsuma M, Furumoto T, Yanagisawa S, Izui K (2005) The ubiquitin-proteasome pathway is involved in rapid degradation of phosphoenolpyruvate carboxylase kinase for C4 photosynthesis. Plant Cell Physiol 46:389–398CrossRefPubMedGoogle Scholar
  2. Araus JL, Bort J, Brown RH, Bassett CL, Cortadellas N (1993) Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthesis gas-exchange characteristics in ears of Triticum durum Desf. Planta 191:507–514CrossRefGoogle Scholar
  3. Bläsing OE, Westhoff P, Svensson P (2000) Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. J Biol Chem 275:27917–27923PubMedGoogle Scholar
  4. Blonde JD, Plaxton WC (2003) Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds. J Biol Chem 278:11867–11873PubMedCrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Chollet R, Vidal J, O’leary MH (1996) Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants. Annu Rev Plant Physiol Plant Mol Biol 47:273–298CrossRefPubMedGoogle Scholar
  7. Cushman JC, Bohnert HJ (1999) Crassulacean acid metabolism: molecular genetics. Annu Rev Plant Physiol Plant Mol Biol 50:305–332CrossRefPubMedGoogle Scholar
  8. Engelmann S, Bläsing OE, Westhoff P, Svensson P (2002) Serine 774 and amino acids 296 to 437 comprise the major C4 determinants of the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia. FEBS Lett 524:11–14CrossRefPubMedGoogle Scholar
  9. Ernst K, Westhoff P (1997) The phosphoenolpyruvate carboxylase (ppc) gene family of Flaveria trinervia (C4) and F. pringlei (C3): molecular characterization and expression analysis of the ppcB and ppcC genes. Plant Mol Biol 34:427–443CrossRefPubMedGoogle Scholar
  10. Gehrig H, Faist K, Kluge M (1998) Identification of phosphoenolpyruvate carboxylase isoforms in leaf, stem and roots of the obligate CAM plant Vanilla planifolia Salib. (Orchidaceae): a physiological and molecular approach. Plant Mol Biol 38:1215–1223PubMedCrossRefGoogle Scholar
  11. González MC, Osuna L, Echevarría C, Vidal J, Cejudo FJ (1998) Expression and localization of phosphoenolpyruvate carboxylase in developing and germinating wheat grains. Plant Physiol 116:1249–1258CrossRefPubMedGoogle Scholar
  12. González MC, Echevarría C, Vidal J, Cejudo FJ (2002) Isolation and characterization of a wheat phosphoenolpyruvate carboxylase gene. Modelling of the encoded protein. Plant Sci 162:233–238CrossRefGoogle Scholar
  13. González MC, Sánchez R, Cejudo FJ (2003) Abiotic stresses affecting water balance induce phosphoenolpyruvate carboxylase expression in roots of wheat seedlings. Planta 216:985–992PubMedGoogle Scholar
  14. Hata S, Izui K, Kouchi H (1998) Expression of a soybean-enhanced phosphoenolpyruvate carboxylase gene that shows striking identity to another gene for a house-keeping isoform. Plant J 13:267–273CrossRefPubMedGoogle Scholar
  15. Izui K, Matsumura H, Furumoto T, Kai Y (2004) Phosphoenolpyruvate carboxylase: a new era of structural biology. Annu Rev Plant Biol 55:69–84CrossRefPubMedGoogle Scholar
  16. Jones JB (1982) Hydroponics: its history and use in plant nutrition studies. J Plant Nutr 5:1003–1030CrossRefGoogle Scholar
  17. Kai Y, Matsumara H, Inoue T, Terada K, Nagara Y, Yoshinaga T, Kihara A, Tsumura K, Izui K (1999) Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition. Proc Natl Acad Sci USA 96:823–828PubMedCrossRefGoogle Scholar
  18. Kawamura T, Shigesada K, Yanagisawa S, Izui K (1990) Phosphoenolpyruvate carboxylase prevalent in maize roots: isolation of a cDNA clone and its use for analysis of the gene and gene expression. Biochem J 107:165–168Google Scholar
  19. Lepiniec L, Keryer E, Philippe H, Gadal P, Cretin C (1993) Sorghum phosphoenolpyruvate carboxylase gene family: structure, function and molecular evolution. Plant Mol Biol 21:487–502CrossRefPubMedGoogle Scholar
  20. Macnicol PK, Raymond P (1998) Role of phosphoenolpyruvate carboxylase in malate production by the developing barley aleurone layer. Physiol Plant 103:132–138CrossRefGoogle Scholar
  21. Mamedov TG, Moellering ER, Chollet R (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga Chlamydomonas reinhardtii. Plant J 42:832–843CrossRefPubMedGoogle Scholar
  22. Martinoia E., Rentsch D. 1994. Malate compartmentation-responses to a complex metabolism. Annu Rev Plant Physiol Plant Mol Biol 45:447–467Google Scholar
  23. MatsumuraH, Xie Y, Shirakata S, Inoue T, Yoshinaga T, Ueno Y, Izui K, Kai Y (2002) Crystal structure of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylase. Structure 10:1721–1730CrossRefPubMedGoogle Scholar
  24. Miyao M, Fukayama H (2003) Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants. Arch Biochem Biophys 414:197–203PubMedGoogle Scholar
  25. Nimmo GA, Nimmo HG, Hamilton ID, Fewson CA, Wilkins MB (1986) Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi. Biochem J 239:213–220PubMedGoogle Scholar
  26. Nimmo HG (2003) Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plants. Arch Biochem Biophys 414:189–196PubMedGoogle Scholar
  27. Podestá FE, Plaxton WC (1994) Regulation of cytosolic carbon metabolism in germinating Ricinus communis cotyledons. II. Properties of phosphoenolpyruvate carboxylase and cytosolic pyruvate kinase associated with the regulation of glycolysis and nitrogen assimilation. Planta 194:381–387Google Scholar
  28. Ryan PR, Delhaize E, Jones DL (2001) Function and mechanism of organic anion exudation from plant roots. Annu Rev Plant Physiol Plant Mol Biol 52:527–560CrossRefPubMedGoogle Scholar
  29. Sánchez R, Cejudo FJ (2003) Identification and expression analysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice. Plant Physiol 132:949–957CrossRefPubMedGoogle Scholar
  30. Sangwan RS, Singh N, Plaxton WC (1992) Phosphoenolpyruvate carboxylase activity and concentration in the endosperm of developing and germinating castor oil seeds. Plant Physiol 99:445–449PubMedGoogle Scholar
  31. Schulz M, Klockenbring T, Hunte C, Schnabl H (1993) Involvement of ubiquitin in phosphoenolpyruvate carboxylase degradation. Bot Acta 106:143–145Google Scholar
  32. Sullivan S, Jenkins GI, Nimmo HG (2004) Roots, cycles and leaves. Expression of the phosphoenolpyruvate carboxylase kinase gene family in soybean. Plant Physiol 135:2078–2087CrossRefPubMedGoogle Scholar
  33. Vidal J, Chollet R (1997) Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase. Trends Plant Sci 2:230–237CrossRefGoogle Scholar
  34. Zhang X-Q, Bin L, Chollet R (1995) In vivo regulatory phosphorylation of soybean nodule phosphoenolpyruvate carboxylase. Plant Physiol 108:1561–1568CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Rosario Sánchez
    • 1
    • 2
  • Amando Flores
    • 1
  • Francisco Javier Cejudo
    • 1
  1. 1.Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la CartujaUniversidad de Sevilla-CSICSevillaSpain
  2. 2.Department of Plant and Microbial BiologyUniversity of CaliforniaBerkeleyUSA

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