A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: identification of AtRALF1 peptide.
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.
Haruta
Miyoshi
M
UW Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706, USA.
Monshausen
Gabriele
G
Gilroy
Simon
S
Sussman
Michael R
MR
D000331:Aequorin; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000737:chemistry* / Q000166:cytology / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D002118:Calcium / Q000737:chemistry* / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology*; D003593:Cytoplasm / Q000737:chemistry / Q000502:physiology; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D036361:Peptide Hormones / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D037401:Scyphozoa; D036226:Seedlings / Q000737:chemistry* / Q000166:cytology / Q000254:growth & development; D015398:Signal Transduction / Q000502:physiology*
D029681:Arabidopsis Proteins; D008024:Ligands; D008164:Luminescent Proteins; D036361:Peptide Hormones; D010455:Peptides; C529880:RALF1 protein, Arabidopsis; D000331:Aequorin; D002118:Calcium