Calmodulin Binds to Extracellular Sites on the Plasma Membrane of Plant Cells and Elicits a Rise in Intracellular Calcium Concentration

Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca²⁺ fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca²⁺ levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom.Calmodulin (CaM)² is a conserved multifunctional calcium sensor that mediates intracellular Ca²⁺ signaling and regulates diverse cellular processes by interacting with calmodulin-binding proteins (1–3). Interestingly, in both animals and plants, CaM may also act as an extracellular agent to regulate physiological events (4). Consistent with this notion, extracellular CaM has been detected within the cell walls of a broad range of plant species (4, 5).Functional studies have established that exogenously applied CaM can stimulate the proliferation of suspension-cultured plant cells (6) as well as affect intracellular activities of heterotrimeric G proteins and phospholipases in protoplasts (7, 8). Based on these findings, it has been proposed that, in plants, extracellular CaM may function as a signaling agent involved in the regulation of cell growth and development (4). However, as a 17-kDa hydrophilic protein, exogenously applied CaM could well be retrieved from the apoplasmic space and then exert its effects on components within the cytoplasm. Evidence against this hypothesis was provided by studies with Arabidopsis thaliana suspension-cultured cells in which it was shown that 24 h of incubation in exogenous CaM did not result in protein uptake or degradation (4).To exert an effect from the apoplasm, it would seem logical to assume that a protein(s) within the plant plasma membrane would have a CaM-binding site exposed to the apoplasm. Although a number of studies have addressed the molecular mechanism(s) by which extracellular CaM might act as a signal (6, 9) and attempts have been made to identify extracellular CaM-binding proteins (4, 6), currently there is no direct evidence in support of the hypothesis that specific CaM-binding sites exist at the surface of plant cells.To address this question, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection (10–13) at the surface of plant cells. These nanoparticles have several advantages over conventional fluorophores for light microscopic imaging, including their higher brightness and photostability (14, 15). In addition, because of their electron dense nature, QDs can be used for single labeling studies at the transmission electron microscope level (16, 17). Using this QD-CaM system, we demonstrate that QD-CaM binds selectively to sites on the outer surface of the plant plasma membrane. We also show by three independent methods that applied CaM can modulate Ca²⁺ fluxes across the plasma membrane, leading to alterations in cytoplasmic Ca²⁺ status. These findings support the hypothesis that, in plants, apoplasmic CaM can act as a signaling agent.