Evidence for multiple receptors mediating RALF-triggered Ca²⁺ signaling and proton pump inhibition

Acidification of the apoplastic space facilitates cell wall loosening and is therefore a key step in cell expansion. PSY1 is a growth-promoting secreted tyrosine-sulfated glycopeptide whose receptor directly phosphorylates and activates the plasma membrane H⁺-ATPase, which results in acidification and initiates cellular expansion. Although the mechanism is not clear, the Rapid Alkalinization Factor (RALF) family of small, secreted peptides inhibits the plasma membrane H⁺-ATPase, leading to alkalinization of the apoplastic space and reduced growth. Here we show that treating Arabidopsis thaliana roots with PSY1 induced the transcription of genes encoding the RALF peptides RALF33 and RALFL36. A rapid burst of intracellular Ca²⁺ preceded apoplastic alkalinization in roots triggered by RALFs, with peptide-specific signatures. Ca²⁺ channel blockers abolished RALF-induced alkalinization, indicating that the Ca²⁺ signal is an obligatory part of the response and that it precedes alkalinization. As expected, fer mutants deficient in the RALF receptor FERONIA did not respond to RALF33. However, we detected both Ca²⁺ and H⁺ signatures in fer mutants upon treatment with RALFL36. Our results suggest that different RALF peptides induce extracellular alkalinization by distinct mechanisms that may involve different receptors.