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

Miyoshi Haruta, Gabriele Brigitte Monshausen, Simon Gilroy, Michael R. Sussman

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

Abstract

Transient increases in the cytoplasmic Ca2+ 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 Ca2+ singling are known to date. To identify endogenous cell signaling peptides regulating cytoplasmic Ca2+ signaling, Arabidopsis seedlings expressing aequorin were used for an in vivo luminescence assay for Ca2+ 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 Ca2+ level increased and reached its maximum. Prior treatment with a Ca2+ chelator or inhibitor of IP3-dependent signaling partially suppressed the AtRALF1-induced Ca2+ concentration increase, indicating the likely involvement of Ca2+ influx across the plasma membrane as well as release of Ca2+ from intracellular reserves. Ca2+ imaging using seedlings expressing the FRET-based Ca2+ 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 Ca2+ levels as reported by aequorin. Our data support a model in which the RALF peptide mediates Ca2+-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.

Original languageEnglish (US)
Pages (from-to)6311-6321
Number of pages11
JournalBiochemistry
Volume47
Issue number24
DOIs
StatePublished - Jun 17 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry

Fingerprint Dive into the research topics of 'A cytoplasmic Ca<sup>2+</sup> functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: Identification of AtRALF1 peptide'. Together they form a unique fingerprint.

Cite this