A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: identification of AtRALF1 peptide

Biochemistry. 2008 Jun 17;47(24):6311-21. doi: 10.1021/bi8001488. Epub 2008 May 22.


Transient increases in the cytoplasmic Ca(2+) concentration are key events that initiate many cellular signaling pathways in response to developmental and environmental cues in plants; however, only a few extracellular mediators regulating cytoplasmic Ca(2+) singling are known to date. To identify endogenous cell signaling peptides regulating cytoplasmic Ca(2+) signaling, Arabidopsis seedlings expressing aequorin were used for an in vivo luminescence assay for Ca(2+) changes. These seedlings were challenged with fractions derived from plant extracts. Multiple heat-stable, protease-sensitive peaks of calcium elevating activity were observed after fractionation of these extracts by high-performance liquid chromatography. Tandem mass spectrometry identified the predominant active molecule isolated by a series of such chromatographic separations as a 49-amino acid polypeptide, AtRALF1 (the rapid alkalinization factor protein family). Within 40 s of treatment with nanomolar concentrations of the natural or synthetic version of the peptides, the cytoplasmic Ca(2+) level increased and reached its maximum. Prior treatment with a Ca(2+) chelator or inhibitor of IP 3-dependent signaling partially suppressed the AtRALF1-induced Ca(2+) concentration increase, indicating the likely involvement of Ca(2+) influx across the plasma membrane as well as release of Ca(2+) from intracellular reserves. Ca(2+) imaging using seedlings expressing the FRET-based Ca(2+) sensor yellow cameleon (YC) 3.6 showed that AtRALF1 could induce an elevation in Ca(2+) concentration in the surface cells of the root consistent with the very rapid effects of addition of AtRALF1 on Ca(2+) levels as reported by aequorin. Our data support a model in which the RALF peptide mediates Ca(2+)-dependent signaling events through a cell surface receptor, where it may play a role in eliciting events linked to stress responses or the modulation of growth.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aequorin
  • Amino Acid Sequence
  • Animals
  • Arabidopsis / chemistry*
  • Arabidopsis / cytology
  • Arabidopsis / growth & development
  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / isolation & purification
  • Arabidopsis Proteins / physiology
  • Calcium / chemistry*
  • Calcium / physiology
  • Calcium Signaling / physiology*
  • Cytoplasm / chemistry
  • Cytoplasm / physiology
  • Ligands
  • Luminescent Proteins / metabolism
  • Luminescent Proteins / physiology
  • Molecular Sequence Data
  • Peptide Hormones / chemistry*
  • Peptide Hormones / isolation & purification
  • Peptide Hormones / physiology
  • Peptides / chemistry*
  • Peptides / isolation & purification
  • Peptides / physiology
  • Scyphozoa
  • Seedlings / chemistry*
  • Seedlings / cytology
  • Seedlings / growth & development
  • Signal Transduction / physiology*


  • Arabidopsis Proteins
  • Ligands
  • Luminescent Proteins
  • Peptide Hormones
  • Peptides
  • RALF1 protein, Arabidopsis
  • Aequorin
  • Calcium