In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays
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 wordsPEPC Phosphoenolpyruvate carboxylase C4 photosynthesis Protein phosphorylation Phosphopeptide antibody Phosphonopeptide antibody Synthetic peptide Immunodetection Zea mays Flaveria bidentis
We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of the manuscript.
- 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
- 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