The Calmodulin-Like Protein AtCML38 Interacts with the AtRALF1 Peptide and the Interaction is Essential for the Inhibition of Root Growth

AtRALF1 is a small secreted peptide hormone that inhibits root growth and has a role in cell elongation. RALF peptides are known to induce a MAP kinase and ion fluxes including extra and intracellular Ca²⁺ mobilization, however their molecular mechanism is still poorly understood. Calmodulin proteins, although well-characterized as intracellular Ca²⁺ sensors, have also been reported as extracellular, secreted proteins in several species including Arabidopsis. Herein we report the identification of the AtCML38, a calmodulin-like protein from Arabidopsis, as an AtRALF1-interacting protein using the yeast two-hybrid system. AtCML38 has been implicated in Ca²⁺-based development and stress responses. Semiquantitative RT-PCR analysis showed co-expression of AtCML38 and AtRALF1 genes in roots of Arabidopsis seedlings. Using bimolecular fluorescence complementation assay we were able to show that AtCML38 interacts with AtRALF1 in the apoplast of Nicotiana benthamiana leaves. Pull-down assays indicated that this interaction occurred in a Ca²⁺- and pH-dependent manner. Expression of the AtRALF1 gene under the control of the strong and constitutive promoter 35S resulted in semi-dwarf plants with pronounced reduction in the root growth. When 35S:AtRALF1 semi-dwarf plants were crossed with a T-DNA insertion mutant defective in the AtCML38 protein, plants with wild-type phenotype, normal root growth and with high accumulation of AtRALF1 peptide were recovered. Our results show that AtCML38 interacts with AtRALF1 peptide and suggest that the interaction is essential for the root growth inhibition caused by the AtRALF1 peptide.