In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays

  • Yoshihisa UenoEmail author
  • Kumiko Yoshizawa-Kumagaye
  • Junji Emura
  • Tomoko Urabe
  • Taku Yoshiya
  • Tsuyoshi Furumoto
  • Katsura Izui
Part of the Methods in Molecular Biology book series (MIMB, volume 2072)


Phosphoenolpyruvate carboxylases (PEPCs), mostly known as the enzymes responsible for the initial CO2 fixation during C4 photosynthesis, are regulated by reversible phosphorylation in vascular plants. The phosphorylation site on a PEPC molecule is conserved not only among isoforms but also across plant species. An anti-phosphopeptide antibody is a common and powerful tool for detecting phosphorylated target proteins with high specificity. We generated two antibodies, one against a peptide containing a phosphoserine (phosphopeptide) and the other against a peptide containing a phosphoserine mimetic, (S)-2-amino-4-phosphonobutyric acid (phosphonopeptide). The amino acid sequence of the peptide was taken from the site around the phosphorylation site near the N-terminal region of the maize C4-isoform of PEPC. The former antibodies detected almost specifically the phosphorylated C4-isoform of PEPC, whereas the latter antibodies had a broader specificity for the phosphorylated PEPC in various plant species. The following procedures are described herein: (1) preparation of the phosphopeptide and phosphonopeptide; (2) preparation and purification of rabbit antibodies; (3) preparation of cell extracts from leaves for analyses of PEPC phosphorylation with antibodies; and (4) characterization of the obtained antibodies. Finally, (5) two cases involving the application of these antibodies are presented.

Key words

PEPC Phosphoenolpyruvate carboxylase C4 photosynthesis Protein phosphorylation Phosphopeptide antibody Phosphonopeptide antibody Synthetic peptide Immunodetection Zea mays Flaveria bidentis 



We thank Edanz Group ( for editing a draft of the manuscript.


  1. 1.
    Chollet R, Vidal J, O’Leary MH (1996) Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants. Annu Rev Plant Physiol Plant Mol Biol 47:273–298CrossRefGoogle Scholar
  2. 2.
    Izui K, Matsumura H, Furumoto T et al (2004) Phosphoenolpyruvate carboxylase: a new era of structural biology. Annu Rev Plant Biol 55:69–84CrossRefGoogle Scholar
  3. 3.
    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 436:15–34CrossRefGoogle Scholar
  4. 4.
    Vidal J, Chollet R (1997) Regulatory phosphorylation of C4 PEP carboxylase. Trends Plant Sci 2:230–237CrossRefGoogle Scholar
  5. 5.
    Hartwell J, Gill A, Nimmo GA et al (1999) Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression. Plant J 20:333–342CrossRefGoogle Scholar
  6. 6.
    Saze H, Ueno Y, Hisabori T et al (2001) Thioredoxin-mediated reductive activation of a protein kinase for the regulatory phosphorylation of C4-form phosphoenolpyruvate carboxylase from maize. Plant Cell Physiol 42:1295–1302CrossRefGoogle Scholar
  7. 7.
    Aldous SH, Weise SE, Sharkey TD et al (2014) Evolution of the phosphoenolpyruvate carboxylase protein kinase family in C3 and C4 Flaveria spp. Plant Physiol 165(3):1076–1091CrossRefGoogle Scholar
  8. 8.
    Dong L-Y, Ermolova NV, Chollet R (2001) Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosphoenolpyruvate carboxylase. Planta 213:379–389CrossRefGoogle Scholar
  9. 9.
    Ueno Y, Hata S, Izui K (1997) Regulatory phosphorylation of plant phosphoenolpyruvate carboxylase: role of a conserved basic residue upstream of the phosphorylation site. FEBS Lett 417:57–60CrossRefGoogle Scholar
  10. 10.
    Takahashi-Terada A, Kotera M, Ohshima K et al (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 280:11798–11806CrossRefGoogle Scholar
  11. 11.
    Ueno Y, Imanari E, Emura J et al (2000) Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide. Plant J 21:17–26CrossRefGoogle Scholar
  12. 12.
    Fukayama H, Hatch MD, Tamai T et al (2003) Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosynth Res 77:227–239CrossRefGoogle Scholar
  13. 13.
    Fukayama H, Tamai T, Taniguchi Y et al (2006) Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice. Plant J 47:258–268CrossRefGoogle Scholar
  14. 14.
    Taniguchi Y, Ohkawa H, Masumoto C et al (2008) Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice. J Exp Bot 59:1799–1809CrossRefGoogle Scholar
  15. 15.
    Furumoto T, Izui K, Quinn V et al (2007) Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis. Plant Physiol 144:1936–1945CrossRefGoogle Scholar
  16. 16.
    Tazoe Y, Hanba YT, Furumoto T et al (2008) Relationships between quantum yield for CO2 assimilation, activity of key enzymes and CO2 leakiness in Amaranthus cruentus, a C4 dicot, grown in high or low light. Plant Cell Physiol 49:19–29CrossRefGoogle Scholar
  17. 17.
    Avasthi UK, Izui K, Raghavendra AS (2011) Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels. J Exp Bot 62:1017–1026CrossRefGoogle Scholar
  18. 18.
    Fukayama H, Fujiwara N, Hatanaka T et al (2014) Nocturnal phosphorylation of phosphoenolpyruvate carboxylase in the leaves of hygrophytic C3 monocots. Biosci Biotechnol Biochem 78:609–613CrossRefGoogle Scholar
  19. 19.
    Wakamiya T, Togashi R, Nishida T et al (1997) Synthetic study of phosphopeptides related to heat shock protein HSP27. Bioorg Med Chem 5:135–145CrossRefGoogle Scholar
  20. 20.
    Higashimoto Y, Saito S, Tong XH et al (2000) Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents. J Biol Chem 275:23199–23203CrossRefGoogle Scholar
  21. 21.
    Tong G, Perich JW, Johns RB (1992) The improved synthesis of Boc-Abu(PO3Me2)-OH and its use for the facile synthesis of Glu-Abu(P)-Leu. Aust J Chem 45:1225–1240CrossRefGoogle Scholar
  22. 22.
    Duff SM, Lepiniec L, Crétin C et al (1993) An engineered change in the L-malate sensitivity of a site-directed mutant of sorghum phosphoenolpyruvate carboxylase: the effect of sequential mutagenesis and S-carboxymethylation at position 8. Arch Biochem Biophys 306:272–276CrossRefGoogle Scholar
  23. 23.
    Dong LY, Masuda T, Kawamura T et al (1998) Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays: comparison with the C4-form enzyme. Plant Cell Physiol 39:865–873CrossRefGoogle Scholar
  24. 24.
    Kawamura T, Shigesada K, Toh H et al (1992) Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function. J Biochem 112:147–154CrossRefGoogle Scholar
  25. 25.
    Wang YH, Chollet R (1993) Partial purification and characterization of phosphoenolpyruvate carboxylase protein-serine kinase from illuminated maize leaves. Arch Biochem Biophys 304:496–502CrossRefGoogle Scholar
  26. 26.
    Tsuchida Y, Furumoto T, Izumida A et al (2001) Phosphoenolpyruvate carboxylase kinase involved in C4 photosynthesis in Flaveria trinervia: cDNA cloning and characterization. FEBS Lett 507:318–332CrossRefGoogle Scholar
  27. 27.
    Ogawa N, Okumura S, Izui K (1992) Ca2+-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize. FEBS Lett 302:86–88CrossRefGoogle Scholar
  28. 28.
    Nagamatsu H, Sakagami A, Yamazaki Y, et al (2010) Development of a highly efficient method for extraction of enzyme proteins from plant materials by the use of skim milk as an assisting agent: a case of PEPC from Eleocharis vivipara. Memoirs of the Faculty of BOST of Kinki University 25:7–16 (in Japanese with English summary)Google Scholar
  29. 29.
    Ogawa N, Kai T, Yabuta N et al (1997) Phosphoenolpyruvate carboxylase of maize leaves: an improved method for purification and reduction of the inhibitory effect of malate by ethylene glycol and bicarbonate. Plant Cell Physiol 38:76–80CrossRefGoogle Scholar
  30. 30.
    Terada K, Kai T, Okuno S et al (1990) Maize leaf phosphoenolpyruvate carboxylase: phosphorylation of Ser15 with a mammalian cyclic AMP-dependent protein kinase diminishes sensitivity to inhibition by malate. FEBS Lett 259:241–244CrossRefGoogle Scholar
  31. 31.
    Jiao JA, Chollet R (1990) Regulatory phosphorylation of Serine-15 in maize phosphoenolpyruvate carboxylase by a C4-leaf protein-serine kinase. Arch Biochem Biophys 283:300–305CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Yoshihisa Ueno
    • 1
    Email author
  • Kumiko Yoshizawa-Kumagaye
    • 2
  • Junji Emura
    • 2
  • Tomoko Urabe
    • 2
  • Taku Yoshiya
    • 2
  • Tsuyoshi Furumoto
    • 1
  • Katsura Izui
    • 3
  1. 1.Department of AgricultureRyukoku UniversityShigaJapan
  2. 2.Peptide Institute, Inc.OsakaJapan
  3. 3.Institute of Advanced Technology, Kindai UniversityWakayamaJapan

Personalised recommendations