, Volume 244, Issue 4, pp 901–913 | Cite as

Phosphoenolpyruvate carboxylase (PEPC) and PEPC-kinase (PEPC-k) isoenzymes in Arabidopsis thaliana: role in control and abiotic stress conditions

  • Ana B. Feria
  • Nadja Bosch
  • Alfonso Sánchez
  • Ana I. Nieto-Ingelmo
  • Clara de la Osa
  • Cristina Echevarría
  • Sofía García-Mauriño
  • Jose Antonio Monreal
Original Article


Main conclusion

Arabidopsis ppc3 mutant has a growth-arrest phenotype and is affected in phosphate- and salt-stress responses, showing that this protein is crucial under control or stress conditions.

Phosphoenolpyruvate carboxylase (PEPC) and its dedicated kinase (PEPC-k) are ubiquitous plant proteins implicated in many physiological processes. This work investigates specific roles for the three plant-type PEPC (PTPC) and the two PEPC-k isoenzymes in Arabidopsis thaliana. The lack of any of the PEPC isoenzymes reduced growth parameters under optimal growth conditions. PEPC activity was decreased in shoots and roots of ppc2 and ppc3 mutants, respectively. Phosphate starvation increased the expression of all PTPC and PPCK genes in shoots, but only PPC3 and PPCK2 in roots. The absence of any of these two proteins was not compensated by other isoforms in roots. The effect of salt stress on PTPC and PPCK expression was modest in shoots, but PPC3 was markedly increased in roots. Interestingly, both stresses decreased root growth in each of the mutants except for ppc3. This mutant had a stressed phenotype in control conditions (reduced root growth and high level of stress molecular markers), but was unaffected in their response to high salinity. Salt stress increased PEPC activity, its phosphorylation state, and L-malate content in roots, all these responses were abolished in the ppc3 mutant. Our results highlight the importance of the PPC3 isoenzyme for the normal development of plants and for root responses to stress.


Anaplerotic function Phosphate starvation Protein kinase Salt stress 



Phosphoenolpyruvate carboxylase


Phosphoenolpyruvate carboxylase kinase


Plant-type phosphoenolpyruvate carboxylase


Bacterial-type phosphoenolpyruvate carboxylase


Root system architecture





We thank Prof. Hugh Nimmo and Dr. Allan James from the University of Glasgow for discussion and advice. This research was supported by Spanish Ministerio de Economía y Competitividad (AGL2012-35708) and by Junta de Andalucía (P12-FQM-489 and PAI group BIO298).

Supplementary material

425_2016_2556_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 14 kb)
425_2016_2556_MOESM2_ESM.docx (12 kb)
Supplementary material 2 (DOCX 12 kb)


  1. Amzallag GN, Lerner HR, Poljakoff-Mayber A (1990) Exogenous ABA as a modulator of the response of Sorghum to high salinity. J Exp Bot 41:1529–1534CrossRefGoogle Scholar
  2. Arias-Baldrich C, Bosch N, Begines D, Feria AB, Monreal JA, García-Mauriño S (2015) Proline synthesis in barley under iron deficiency and salinity. J Plant Physiol 183:121–129CrossRefPubMedGoogle Scholar
  3. Armengaud P, Zambaux K, Hills A, Sulpice R, Pattison RJ, Blatt MR, Amtmann A (2009) EZ-Rhizo: integrated software for fast and accurate measurement of root system architecture. Plant J 57:945–949CrossRefPubMedGoogle Scholar
  4. Bates IS, Waldren RP, Teare JD (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  5. Beers RF, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195(1):133–140PubMedGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  7. Chen Z-H, Nimmo GA, Jenkins GI, Nimmo HG (2007) BHLH32 modulates several biochemical and morphological processes that respond to Pi starvation in Arabidopsis. Biochem J 405:191–198CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen Z-H, Jenkins GI, Nimmo HG (2008a) Identification of an F-box protein that negatively regulates Pi starvation responses. Plant Cell Physiol 49:1902–1906CrossRefPubMedGoogle Scholar
  9. Chen Z-H, Jenkins GI, Nimmo HG (2008b) pH and carbon supply control the expression of phosphoenolpyruvate carboxylase kinase genes in Arabidopsis thaliana. Plant Cell Environ 31:1844–1850CrossRefPubMedGoogle Scholar
  10. Chen M, Tang Y, Zhang J, Yang M, Xu Y (2010) RNA interference-based suppression of phosphoenolpyruvate carboxylase results in susceptibility of canola to osmotic stress. J Integr Plant Biol 52:585–592CrossRefPubMedGoogle Scholar
  11. 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
  12. Doubnerova V, Ryslava H (2011) What can enzymes of C4 photosynthesis do for C3 plants under stress? Plant Sci 180:575–583CrossRefPubMedGoogle Scholar
  13. Echevarría C, Vidal J (2003) The unique phosphoenolpyruvate carboxylase kinase. Plant Physiol Biochem 41:541–547CrossRefGoogle Scholar
  14. Echevarría C, Vidal J, Jiao JA, Chollet R (1990) Reversible light activation of phosphoenolpyruvate carboxylase protein-serine kinase in maize leaves. FEBS Lett 275:25–28CrossRefPubMedGoogle Scholar
  15. Echevarría C, Pacquit V, Bakrim N, Osuna L, Delgado B, Arrio-Dupont M, Vidal J (1994) The effect of pH on the covalent and metabolic control of C4 phosphoenolpyruvate carboxylase from Sorghum leaf. Arch Biochem Bioph 315:425–430CrossRefGoogle Scholar
  16. Echevarría C, García-Mauriño S, Alvarez R, Soler A, Vidal J (2001) Salt stress increases the Ca2+-independent phosphoenolpyruvate carboxylase kinase activity in Sorghum plants. Planta 214:283–287CrossRefPubMedGoogle Scholar
  17. Fedosejevs ET, Ying S, Park J, Anderson EM, Mullen RT, She Y-M, Plaxton WC (2014) Biochemical and molecular characterization of RcSUS1, a cytosolic sucrose synthase phosphorylated in vivo at serine 11 in developing castor oil seeds. J Biol Chem 289:33412–33424CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fontaine V, Hartwell J, Jenkins GI, Nimmo GA, Nimmo HG (2002) Arabidopsis thaliana contains two phosphoenolpyruvate carboxylase kinase genes with different expression patterns. Plant Cell Environ 25:115–122CrossRefGoogle Scholar
  19. Fukayama H, Tamai T, Taniguchi Y, Sullivan S, Miyao M, Nimmo HG (2006) Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice. Plant J 47:258–268CrossRefPubMedGoogle Scholar
  20. García-Mauriño S, Monreal JA, Alvarez R, Vidal J, Echevarría C (2003) Characterization of salt stress-enhanced phosphoenolpyruvate carboxylase kinase activity in leaves of Sorghum vulgare: independence from osmotic stress, involvement of ion toxicity and significance of dark phosphorylation. Planta 216:648–655PubMedGoogle Scholar
  21. González MC, Sánchez R, Cejudo FJ (2003) Abiotic stresses affecting water balance induces phosphoenolpyruvate carboxylase expression in roots of wheat seedlings. Planta 216:985–992PubMedGoogle Scholar
  22. Gousset-Dupont A, Lebouteiller B, Monreal JA, Echevarría C, Pierre JN, Hodges M, Vidal J (2005) Metabolite and posttranslational control of phosphoenolpyruvate carboxylase from leaves and mesophyll cell protoplasts of Arabidopsis thaliana. Plant Sci 169:1096–1101CrossRefGoogle Scholar
  23. Gregory AL, Hurley BA, Tran HT, Valentine AJ, She YM, Knowles VL, Plaxton WC (2009) In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in phosphate-starved Arabidopsis thaliana. Biochem J 420:57–65CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499CrossRefPubMedGoogle Scholar
  25. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplast: kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198CrossRefPubMedGoogle Scholar
  26. Hu Y, Fromm J, Schmidhalter U (2005) Effect of salinity on tissue architecture in expanding wheat leaves. Planta 220:838–848CrossRefPubMedGoogle Scholar
  27. 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
  28. Julkowska MM, Testerink C (2015) Tuning plant signaling and growth to survive salt. Trends Plant Sci 20:586–594CrossRefPubMedGoogle Scholar
  29. Kandoi D, Mohanty S, Govindjee Tripathy BC (2016) Towards efficient photosynthesis: overexpression of Zea mays phosphenolpyruvate carboxylase in Arabidopsis thaliana. Photosynth Res. doi: 10.1007/s11120-016-0224-3 PubMedGoogle Scholar
  30. Li B, Chollet R (1994) Salt induction and the partial purification/characterization of phosphoenolpyruvate carboxylase protein-serine kinase from an inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum L. Arch Biochem Biophys 314:247–254CrossRefPubMedGoogle Scholar
  31. Liu J, Zhu J-K (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114:591–596CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lowry OH, Passoneau JV (1972) A flexible system of enzymatic analysis. Academic press, London, pp 201–204Google Scholar
  33. Monreal JA, Feria AB, Vinardell JM, Vidal J, Echevarría C, García- Mauriño S (2007a) ABA modulates the degradation of phosphoenolpyruvate carboxylase kinase in Sorghum leaves. FEBS Lett 581:3468–3472CrossRefPubMedGoogle Scholar
  34. Monreal JA, López-Baena FJ, Vidal J, Echevarría C, García-Mauriño S (2007b) Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of Sorghum vulgare. Planta 225:801–812CrossRefPubMedGoogle Scholar
  35. Monreal JA, Lopez-Baena FJ, Vidal J, Echevarría C, García-Mauriño S (2010a) Involvement of phospholipase d and phosphatidic acid in the light-dependent up-regulation of Sorghum leaf phosphoenolpyruvate carboxylase-kinase. J Exp Bot 61:2819–2827CrossRefPubMedPubMedCentralGoogle Scholar
  36. Monreal JA, McLoughlin F, Echevarría C, García-Mauriño S, Testerink C (2010b) Phosphoenolpyruvate carboxylase from C4 leaves is selectively targeted for inhibition by anionic phospholipids. Plant Physiol 152:634–638CrossRefPubMedPubMedCentralGoogle Scholar
  37. Monreal JA, Arias-Baldrich C, Pérez-Montaño F, Gandullo J, Echevarría C, García-Mauriño S (2013a) Factors involved in the rise of phosphoenolpyruvate carboxylase-kinase activity caused by salinity in Sorghum leaves. Planta 237:1401–1413CrossRefPubMedGoogle Scholar
  38. Monreal JA, Arias-Baldrich C, Tossi V, Feria AB, Rubio-Casal A, García-Mata C, Lamattina L, García-Mauriño S (2013b) Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in Sorghum responses to salinity. Planta 238:859–869CrossRefPubMedGoogle Scholar
  39. Morcuende R, Bari R, Gibon Y, Zheng W, Pant BD, Bläsing O, Usadel B, Czechowski T, Udvardi MK, Stitt M, Scheible W (2007) Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus. Plant, Cell Environ 30:85–112CrossRefGoogle Scholar
  40. Muller R, Morant M, Jarmer H, Nilsson L, Nielsen TH (2007) Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol 143:156–171CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nimmo GA, McNaughton GAL, Fewson CA, Wilkins MB, Nimmo HG (1987) Changes in the kinetic properties and phosphorylation state of phosphoenolpyruvate carboxylase in Zea mays leaves in response to light and dark. FEBS Lett 213:18–22CrossRefGoogle Scholar
  42. Niu YF, Chai RS, Jin GL, Wang H, Tang CX, Zhang YS (2013) Responses of root architecture development to low phosphorus availability: a review. Ann Bot Lond 112:391–408CrossRefGoogle Scholar
  43. O’Leary B, Park J, Plaxton WC (2011) The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J 436:15–34CrossRefPubMedGoogle Scholar
  44. Pacquit V, Giglioli N, Crétin C, Pierre JN, Vidal J, Echevarría C (1995) Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase from Sorghum: an immunological study using specific anti-phosphorylation site-antibodies. Photosynth Res 43:283–288CrossRefPubMedGoogle Scholar
  45. Péret B, Clément M, Nussaume L, Desnos T (2011) Root developmental adaptation to phosphate starvation: better safe than sorry. Trends Plant Sci 16:442–450CrossRefPubMedGoogle Scholar
  46. Popova LP, Stoinova ZG, Maslenkova LT (1995) Involvement of abscisic acid in photosynthetic process in Hordeum vulgare L. during salinity stress. J Plant Growth Regul 14:211–218CrossRefGoogle Scholar
  47. Rubio V, Linhares F, Solano R, Martín AC, Igresias J, Leyva A, Paz-Ares J (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev 15:2122–2133CrossRefPubMedPubMedCentralGoogle Scholar
  48. Ruiz-Ballesta I, Feria AB, Ni H, She Y-M, Plaxton WC, Echevarría C (2014) In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating Sorghum seeds. J Exp Bot 65:443–451CrossRefPubMedGoogle Scholar
  49. Sánchez R, Cejudo FJ (2003) Identification and expression analysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice. Plant Physiol 132:949–957CrossRefPubMedPubMedCentralGoogle Scholar
  50. Sánchez R, Flores A, Cejudo FJ (2006) Arabidopsis phosphoenolpyruvate carboxylase genes encode immunologically unrelated polypeptides and are differentially expressed in response to drought and salt stress. Planta 223:901–909CrossRefPubMedGoogle Scholar
  51. Seki M, Umezawa T, Urano K, Shinozaki K (2007) Regulatory metabolic networks in drought stress responses. Curr Opin Plant Biol 10:296–302CrossRefPubMedGoogle Scholar
  52. Shane MW, Fedosejevs ET, Plaxton WC (2013) Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea. Plant Physiol 161:1634–1644CrossRefPubMedPubMedCentralGoogle Scholar
  53. Shenton M, Fontaine V, Hartwell J, Marsh JT, Jenkins GI, Nimmo HG (2006) Distinct patterns of control and expression amongst members of the PEP carboxylase kinase gene family in C4 plants. Plant J 48:45–53CrossRefPubMedGoogle Scholar
  54. Shi J, Yi K, Liu Y, Xie L, Zhou Z, Chen Y, Hu Z, Zheng T, Liu R, Chen Y, Chen J (2015) Phosphoenolpyruvate carboxylase in Arabidopsis leaves plays a crucial role in carbon and nitrogen metabolism. Plant Physiol 167:671–681CrossRefPubMedPubMedCentralGoogle Scholar
  55. Takahashi-Terada A, Kotera M, Ohshima K, Furumoto T, Matsumura H, Kai Y, Izui K (2005) Maize phosphoenolpyruvate carboxylase. Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation. J Biol Chem 280:11798–11806CrossRefPubMedGoogle Scholar
  56. Taybi T, Patil S, Chollet R, Cushman JC (2000) A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in Crassulacean acid metabolism-induced leaves of the common ice plant. Plant Physiol 123:1471–1481CrossRefPubMedPubMedCentralGoogle Scholar
  57. Taybi T, Nimmo HG, Borland AM (2004) Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of Crassulacean acid metabolism. Plant Physiol 135:587–598CrossRefPubMedPubMedCentralGoogle Scholar
  58. Uhrig RG, O’Leary B, Spang HE, MacDonald JA, She Y, Plaxton WC (2008) Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds. Plant Physiol 146:1346–1357CrossRefPubMedPubMedCentralGoogle Scholar
  59. Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423–447CrossRefGoogle Scholar
  60. Vidal J, Chollet R (1997) Regulatory phosphorylation of C4 PEP carboxylase. Trends Plant Sci 2:230–237CrossRefGoogle Scholar
  61. Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ana B. Feria
    • 1
  • Nadja Bosch
    • 1
  • Alfonso Sánchez
    • 1
  • Ana I. Nieto-Ingelmo
    • 1
  • Clara de la Osa
    • 1
  • Cristina Echevarría
    • 1
  • Sofía García-Mauriño
    • 1
  • Jose Antonio Monreal
    • 1
  1. 1.Departamento de Biología Vegetal y Ecología, Facultad de BiologíaUniversidad de SevillaSevilleSpain

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