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

Abstract

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 Ca 2؉ 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 Ca 2؉ 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 Ca 2ϩ 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 ce...

Calcium at the Cell Wall‐Cytoplast Interface

“…With ion imaging, Shang et al (2005) first showed that extracellular addition of purified cauliflower CaM stimulates Ca 2+ entry into pollen grains. In an extension of that study, Wang et al (2009) report that the quantum dot‐CaM probe also stimulates Ca 2+ influx. In parallel with results noted above, Shang et al (2005) further show that application of a CaM anti‐serum, or the CaM antagonist W‐7, inhibits the increase in intracellular Ca 2+ .…”

Ca2+signals: The versatile decoders of environmental cues

“…Its presence is suggested in other organellar membranes as well (Schiott et al, 2004;Ali et al, 2007). Its extracellular functions are also reported (Chen et al, 2004;Li et al, 2009;Wang et al, 2009). CaM-binding proteins are versatile in nature and include kinases, cytoskeletal proteins, metabolic enzymes, TFs, channels and pumps, and many proteins of unknown function (Bouche et al, 2005;Ikura & Ames, 2006).…”

NaCl-elicited, vacuolar Ca2+ release facilitates prolonged cytosolic Ca2+ signaling in the salt response of Populus euphratica cells

“…The high level of cytosolic Ca 2+ was maintained for several minutes [44]. The prolonged rise in cytosolic Ca 2+ (several minutes or longer) was also observed in response to different stimulus when single wavelength, non-ratiometric dyes were used as Ca 2+ indicator, such as Fluo-3, Calcium Green [58][59][60]. Moreover, cell-and stimulus type-specific intracellular free Ca 2+ signals were reported in Arabidopsis, in which a prolonged elevation (several minutes) of cytosolic Ca 2+ was observed in guard cells [61].…”