Communicating with Calcium

Abstract

All living cells use a network of signal transduction pathways to conduct developmental programs, obtain nutrients, control their metabolism, and cope with their environment. A major challenge for cell biologists is to understand the “language” of these signaling systems. For simplicity, signaling pathways usually have been studied in isolation, with experimenters attempting to define a single pathway through which a given stimulus evokes a response. However, cells are not simple, and for any given stimulus (input), the final response (output) is likely to be the result of complex interaction, or cross-talk, between multiple pathways (Trewavas and Malhó, 1997; Jenkins, 1998). Presumably, this cross-talk evolved as a mechanism to enable a relatively small number of messengers to help cells process a much larger array of potential stimuli in an appropriate fashion. In plant cells, the list of messengers used by signaling pathways includes Ca2+, lipids, pH, and cyclic GMP (cGMP). However, no single messenger has been demonstrated to respond to more stimuli than has cytosolic free Ca2+ ([Ca2+]c). A partial list of stimuli that evoke changes in [Ca2+]c is shown in Table 1. Our knowledge of [Ca2+]c in plant cell signaling has been markedly enhanced by the development of reliable techniques to measure free Ca2+ intracellularly (Read et al., 1993). Early studies (Brownlee and Wood, 1986; Miller and Sanders, 1987; Felle, 1989) with Ca2+-selective microelectrodes yielded consistent estimates of [Ca2+]c. However, electrodes are rather unwieldy and are unable to report detailed spatial information, so they have been largely supplanted by fluorescent dyes and the luminescent protein aequorin (Read et al., 1993). Ca2+-indicating fluorescent dyes usually are microinjected into the cell or sometimes loaded as the free acid at low pH. Their fluorescence yields are sufficient to enable ready imaging of heterogeneities in [Ca2+]c,; for dyes in which an isosbestic point is present in the excitation or emission spectrum, ratiometric images allow quantification of the signal independent of dye concentration. Alternatively, changes in [Ca2+]c can be visualized with the Ca2+-sensitive luminescent protein aequorin. The use of transgenic plants stably expressing apoaequorin— from which aequorin can be spontaneously reconstituted on addition of the luminophore coelenterazine—circumvents the requirement for microinjection, although low-intensity emission of aequorin so far has limited spatial resolution of [Ca2+]c signals to the multicellular level (Knight et al., 1998). Although calcium signals have been implicated in many stimulus–response phenomena, rigorous proof that a [Ca2+]c transient is an essential intermediate has been difficult to obtain. A strong argument for a critical involvement of Ca2+ requires the demonstration not only that a stimulus evokes a change in [Ca2+]c but also that two additional experimental criteria are met. First, a block of the [Ca2+]c transient also should block the downstream response, and second, appropriate Ca2+-sensitive response elements should be present. Meeting these criteria represents a formidable technical challenge, but strong evidence exists to support a central role of [Ca2+]c in a range of events, including phytochrome action (Shacklock et al., 1992; Bowler et al., 1994; Neuhaus et al., 1997), stomatal closure in response to a number of stimuli (MacRobbie, 1997), and tip growth of root hairs, pollen tubes, and algal rhizoids (e.g., Brownlee and Wood, 1986; Wymer et al., 1997; Holdaway-Clarke et al., 1998; Malhó et al., 1998). Metabolism in all cells requires the presence of orthophosphate (Pi) and phosphorylated organic compounds, particularly for cytosolic reactions associated with transduction of free energy. The low solubility product of Ca2+ with Pi would have required the early evolution of mechanisms for maintenance of [Ca2+]c at a level that would certainly be well below the millimolar concentrations that prevail in seawater. Thus, transport systems that export Ca2+ from the cytosol are present in all cells to sustain steady state values of [Ca2+]c in the submicromolar range. This homeostatic mechanism would have been ideal for subsequent evolution of Ca2+-based signaling pathways. Specifically, the elevation of [Ca2+]c by a factor of 10 or 20 can occur more rapidly than would be possible for ions or solutes that are maintained at millimolar levels. Some Physiological Stimuli That Elevate [Ca2+]c in Plant Cells Some Physiological Stimuli That Elevate [Ca2+]c in Plant Cells The chemistry of Ca2+ provides further favorable features for signal transduction. The ability of the ion to coordinate a considerable number (commonly six to eight) of uncharged oxygen atoms enables the evolution of protein conformations in which remote domains can participate in calcium binding (McPhalen et al., 1991). Calcium-induced conformational changes can in fact be exploited to elicit downstream events