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 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.