Use of Mass Spectrometry‐Based Phosphoproteomics to Characterize a Receptor Protein Kinase‐Mediated Signaling Pathway that Negatively Regulates Plant Cell Growth.

Plant cells are immobile and thus, cell expansion plays a critical role in the shape, growth and development of plants. The precise molecular mechanism by which the plasma membrane initiates changes in the cellular growth rate remains elusive. Using metabolic labeling with the stable isotope, 15 N, in Arabidopsis seedlings we performed isotope‐assisted quantitative mass spectrometric measurements of the plasma membrane phosphoproteome following brief treatments with a secreted 49 amino acid long peptide known as RALF (for R apid Al kalinization F actor). This hormone causes alkalinization of the extracellular medium and suppresses cell elongation. Out of ~500‐600 phosphopeptides in the plasma membrane fraction quantified, we found a handful of proteins in which at least one phosphosite was altered. The largest change was observed in phosphosites at the carboxy terminus of the cell surface receptor kinase known as FERONIA and we hypothesized that this was due to autophosphorylation induced by RALF binding to the receptor. A direct peptide‐receptor interaction was supported by specific binding of RALF to the extracellular domain of FERONIA and reduced binding and insensitivity to RALF‐induced growth inhibition in feronia mutants. Phosphoproteome measurements also demonstrated that the RALF‐FERONIA interaction caused phosphorylation of a plasma membrane proton pump at Ser899, mediating the inhibition of proton transport. These results, together with additional reverse genetic analysis of the phosphorylated proteins, reveal a molecular mechanism for RALF‐induced extracellular alkalinization and a signaling pathway that regulates cell expansion.