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 in signaling pathways. The cytosolic free calcium concentration typically is maintained at ∼200 nM (Bush, 1995). However, the Ca2+ content of the cytosol is far higher than this because of the high affinity for Ca2+ to a range of binding proteins. Prominent among these proteins is calmodulin, which, by binding in turn to other proteins, serves as an adaptor of Ca2+ signals. Much of the calcium that enters the cytosol during signaling is rapidly bound. Estimates from neuronal cells suggest that only 0.1 to 1% of an imposed Ca2+ load remains free (Brinley et al., 1977; Gorman and Thomas, 1980), with the rest being absorbed. The tendency for the majority of Ca2+ ions to associate with cytosolic proteins has the effect of markedly reducing the effective diffusion coefficient of Ca2+ in the cytosol (Thomas, 1982). Restricted rates of diffusion in the cytosol underlie the occurrence of localized and spatially distinct patterns in Ca2+ signaling. The presence of intracellular organelles that are able to accumulate Ca2+ effectively contributes to the buffering of a Ca2+ load, tending to dampen Ca2+ signals. Whereas the endoplasmic reticulum (ER), mitochondria, and even chloroplasts are all known to have the capacity to sequester Ca2+ (Bush, 1995), it is vacuoles that, by virtue of their size and capacity for Ca2+ accumulation, represent the most prominent sink for Ca2+ in most cells. The relative contributions of biochemical and organellar buffering have not been estimated in plant cells. However, in the mycelial fungus Neurospora crassa, buffering of Ca2+ is extremely effective, with vacuolar sequestration probably playing the major role (Miller et al., 1990). Ultimately, homeostasis of [Ca2+]c must be achieved by export across the plasma membrane because both biochemical buffering and intracellular sequestration have finite capacities. Hence, a considerable electrochemical potential difference for Ca2+(Δμ̄Ca) exists across all membranes in the cell, with the energized transport of Ca2+ invariably in a direction out of the cytosol. At the plasma membrane, the Ca2+ concentration ratio (inside/outside) is typically of the order of 10-4, and the presence of a cytosolic negative electrical potential difference of approximately yields a of approximately Although the potential difference across the membranes of as the and is likely to be negative than that across the plasma membrane, prevail and energized of Ca2+ from the cytosol. of Ca2+ from the cytosol is by by or a the of Ca2+ into the cytosol is by ion Ca2+ signals can be of as the result of two into and out of the cytosol. In the of the transport systems and are the of the of a Ca2+ However, Ca2+ binding proteins, by virtue of their in as of [Ca2+]c has the role of in [Ca2+]c at the and plasma an of the Ca2+ transport events at these which are the of more detailed Ca2+ across as the membrane, and membrane, be in the or of Ca2+ signals et al., et al., and of Ca2+ in Plant transport systems are as and are Ca2+ that have been localized to the membranes The direction of Ca2+ for is to a plasma membrane that has been but not at a is a Ca2+ that to the vacuolar is a from probably at the vacuolar represent ion through has been at a Ca2+ by vacuolar Ca2+ vacuolar Ca2+ for further and The transport systems that from the cytosol are to critical First, they [Ca2+]c to Ca2+ signals. they load Ca2+ into as the and to be used as for Ca2+ they Ca2+ to organelles to support biochemical For high of Ca2+ in the are required for protein through the pathway et al., et al., A is in addition to their any of these pathways help the of a calcium signal and help define the in calcium is to the signals that control these systems be in the signals that calcium in two systems and has demonstrated that the or of a Ca2+ can signal transduction and et al., 1998; et al., 1998). Thus, the potential signaling of systems in plants must be can in transport of Ca2+ in which a is although in plants this usually requires a of at et al., 1990). The plant to be and et al., The by ability to growth on to a in vacuolar Ca2+ to transport Ca2+ at low concentrations is consistent with studies on with root vacuoles (e.g., and Although it is to that is localized to the plant is evidence for in other as the plasma membrane and 1990). Thus, the of at the to the of that use to ion distinct Ca2+ have been on protein and 1998). of the and the and plasma Ca2+ in cells. and plasma by to the or plasma membrane, to (e.g., by acid and and of plasma by and 1998). have been from including from et al., from et al., 1997), and from et al., is evidence that is in the However, in to the have been for other For of in membrane in both and plasma membrane and plant have been and from et al., et al., and from et al., The plant proteins are from plasma by at membranes other than the plasma membrane, and a with domains at the of the that is in a membrane, on membrane and with an et al., 1993). to be in the vacuolar membrane, on with a from a vacuolar et al., shown to with the as by of in membrane and by of an with a fluorescent protein et al., 1998; et al., The and two in which multiple pathways have been in the In is evidence for both a and a In the is evidence for two of calcium a and is not are by the pathways. is that the Ca2+ pathways are in by to and systems. The Ca2+ is by a and In both and plasma to be by multiple signal transduction including and multiple (e.g., and protein 1997; 1997; and 1998). The can be in response to signals as well as a that the Ca2+ load the and 1998; et al., 1998). In the of is the of at by (e.g., and have to be at a level in a in Ca2+ et al., 1997; et al., 1998). Although the of provides no to likely to ion an is that provides a pathway for Ca2+ in the of and biochemical studies have in a considerable of on the and of at the vacuolar and plasma In with to are into their in cell signaling. At two major of Ca2+ in the plasma membrane 1998). that are relatively with to and a high are known as The is relatively more for a and is known as 1994; and both of ion have been most in it is that in a of other cell and including and et al., et al., and and root cells et al., 1992; et al., A of most is their capacity for by membrane is that the critical factor for during signaling et al., 1995). Thus, a number of including and et al., et al., and et al., evoke membrane probably through of and et al., The in Ca2+ an in [Ca2+]c. However, additional of Ca2+ are likely to be required to of Ca2+ signals by the many (e.g., and organic that have been shown to plant plasma The demonstration that the of is by or with et al., is in with the of the presence of additional Ca2+ pathways into plant cells. In cells, for it is likely that acid Ca2+ by of and and et al., The of by acid and that the of the that underlie the be by protein or At have been at vacuolar membranes and Sanders, of these are by and 1987; et al., the other by cyclic et al., 1995). The of plant of and Sanders, which, with are for of Ca2+ during signaling in cells. of both and into cells has that both have the capacity to that and are in Ca2+ in plants et al., et al., 1998). A of both the and the of cells is their capacity for by [Ca2+]c. This response is to underlie Ca2+ which can be to the of Ca2+ signals and 1995). across the vacuolar membrane of plants are by [Ca2+]c and Sanders, et al., the presence of and of [Ca2+]c which are of The two at the vacuolar membrane are both by membrane et al., 1992; and and Sanders, and the other by membrane and 1994; and and Sanders, The of these is known as the vacuolar and it also is by [Ca2+]c nM and has been that a for and with Ca2+ by the of or both of the as the for This has been as a result of the that the of the is on and that, at values of of Ca2+ from the the of the small et al., However, this to be as a result of the that the of the is markedly enhanced by the presence of cytosolic ions et al., which not present in the of et the of the as an intracellular of Ca2+ in cells, the that other intracellular participate in Ca2+ signaling should not be imaging studies have demonstrated intracellular Ca2+ from of pollen and root in which no is et al., et al., and it is not that Ca2+ have been at A Ca2+ has been in the from and in et al., 1995). by has been demonstrated in from which than the vacuolar and associated with and Sanders, Physiological for both and are studies and have for in stomatal closure et al., et al., et al., of in the cells of by et al., and of pollen during the response et al., is evidence for a role of Ca2+ signaling in et that are by and used a to that evokes of two Although is by a range of to as microinjection, the response is by the but not by is enhanced by suggest considerable in the that a of Ca2+ or that the of the [Ca2+]c signal are critical for the given response. In a et al., stomatal closure shown to be both by and by of The of Ca2+ signal transduction pathways can be into two and downstream Some downstream represent the point for the signaling pathway and ion In cells, in during stomatal closure are by [Ca2+]c and by and 1992; et al., 1995). In the of Ca2+ of the of Ca2+ are by and 1994; and 1995). protein are a of by a with calcium binding are likely to be of Ca2+ signals and in plants and et al., and et al., 1993). have been in the of and In on the other have been with or more (e.g., and et al., 1995). In of the have been et al., The of with the of potential that or protein proteins, has to the that most protein in plants through pathways and 1998). Some to distinct signaling pathways. For in a transient only two of from the distinct for et al., 1998). In are by concentrations of the that Ca2+ signals. Although the of has not been it is that are multiple in both and and The of potential of remains a major on in is a list of potential including and in cells et al., et al., the in root et al., 1995), et al., 1995), and the plasma membrane et al., 1998; et al., et al., and et al., 1998). However, the more difficult is to that a is by a pathway the A role for as of Ca2+ signals also is in cells, which are during stomatal are by but by which et al., 1993). are by and it has been that are in the of this be as a negative mechanism to the cytosol from being by the vacuolar Ca2+ and Sanders, or as a of [Ca2+]c and 1998). A more role for is at by the and that the of a of the from well as neuronal calcium from are to and the is by Ca2+ concentration and is known to elicit changes in [Ca2+]c (Knight et al., 1997), be the Ca2+ in a signal transduction However, it should be that although the is to the changes in free Ca2+ are not and also are by (Knight et al., signaling are likely to of Ca2+ signals. The that plants (e.g., et al., a role for in because Ca2+ by would further The of plant has that they are both Ca2+ and to the et al., 1998). In with the that participate in Ca2+ Ca2+ in pollen and in [Ca2+]c in cells are by et al., et al., In both of these Ca2+ to participate in the of The involvement of Ca2+ in a of stimulus–response pathways in plant cells can be by the a of to stimuli of is a of plant to changes et al., 1997; Malhó et al., 1998; and 1998). features of the Ca2+ signaling are to a single messenger can multiple and of the Ca2+ as well as the with other and signaling pathways that the of the given cell et al., In cells, to Ca2+ signals in and have been The and of a Ca2+ signal in for of et al., in the of Ca2+ cell and 1995). a of et that the of Ca2+ can the of a factor of cells, support to the that can be into the of Ca2+ signals and in cells, of can elicit changes in response changes in [Ca2+]c by of response elements et al., The demonstration that a is by Ca2+ that Ca2+ can protein et al., that of Ca2+ signal can is for plant cells. are of single Ca2+ in response to in [Ca2+]c in response to or both vacuolar and plasma membrane Ca2+ (Knight et al., and transient in of in response to et al., (Knight et al., et al., and et al., et al., et al., The Ca2+ signals to downstream is that the of the Ca2+ transient the of the stimulus and the of the response. Malhó et have Ca2+ transient to stimuli and point out that with to and [Ca2+]c signal distinct that be as an of the For the in response to is the is and the of the transient only in cells a of by a of and signal of The of Ca2+ has been shown to on the size of the et al., 1997; Brownlee et al., In the Ca2+ transient with a small elevation a transient on the presence of Ca2+, although they to the and the protein the involvement of mechanisms for The also that required as of the it shown that the from from the the larger transient elevation of Ca2+ from intracellular et al., 1998). In the of Ca2+ from or be by or (Knight et al., 1998). Ca2+ signals well with changes in patterns of and the of However, a more complex to between Ca2+ signals and the downstream in Thus, and can Ca2+ of and with of of the which for an in (Knight et al., suggest that other than Ca2+ are in between the two response pathways. A further in Ca2+ signals in of a response has been demonstrated in in response to and Specifically, phytochrome control of the is by and by the response to by et al., 1998). as to have on the of the in the cell to in the that the to and plant cell respond to a of and stimuli with in [Ca2+]c et al., 1994; et al., et al., et al., 1998; et al., 1998). these represent Ca2+ signals or are the result of of Ca2+ homeostasis requires the demonstration of downstream on the or of the to Ca2+ signals have been in only a in in root occur in response to et al., is by Ca2+ in in et al., in the Ca2+ with growth in pollen In cells, can be by as changes in and et al., et al., The and of Ca2+ have been shown to the stomatal the and of the Ca2+ on the of the imposed Ca2+ concentration. to high concentrations of Ca2+ to in to to nM with a A mechanism for from patterns of Ca2+ has been et al., and that the action of a and a protein in the of would be in the of of proteins to A further mechanism by which Ca2+ signals can and that is in many of plant signal the of the signal to a Ca2+ signals by localized to the of root et al., spatial from the Ca2+ signals in root growth The use of to the or has the presence of of and cytosolic Ca2+ from to et al., 1995). In shown to of but not in et al., 1998). In cells, many Ca2+ signals have been shown to as of at a and as of Ca2+ from intracellular 1995). In are of Ca2+ in The Ca2+ transient in cells as a of that at the and Ca2+ at and Ca2+ from intracellular et al., The of is at to 10 to the of of intracellular Ca2+ in a of systems 1995). Ca2+ have been in other plant although a of intracellular Ca2+ can be in pollen in response to of evidence also exists for the occurrence of a Ca2+ in cells and 1998). The and of this Ca2+ signal by the presence of In cells, Ca2+ of Ca2+ from the The of many higher plant cells by a central be Ca2+ have been in both and pollen tubes, Ca2+ through that but no for the involvement of intracellular Ca2+ that spatially distinct Ca2+ signals from studies of plants and the use of aequorin to in for both vacuolar and plasma membrane Ca2+ are implicated in and Ca2+ signals. In a Ca2+ to be in response to signals et al., additional in which the or of a Ca2+ signal is Ca2+ have been in of pollen et al., Miller et al., 1992; et al., 1997), root and Felle, and 1997; Wymer et al., 1997), and algal rhizoids (Brownlee and Wood, 1986; Brownlee and and et al., Ca2+ at the can levels. The Ca2+ in pollen with growth et al., Miller et al., of the Ca2+ into pollen growth and the Ca2+ (Miller et al., pollen be to localized of by as of Ca2+ at the pollen et al., Ca2+ shown to with in growth and is to that the and of the Ca2+ that growth et al., in growth by in Ca2+ at the pollen However, of Ca2+ at the by an Ca2+-selective that in Ca2+ with the but out of growth and [Ca2+]c. This Ca2+ associated with the of a as the although the of pollen is not particularly at the and Alternatively, the Ca2+ changes in Ca2+ binding to cell during growth et al., The of Ca2+ and pollen growth is not as pollen can Ca2+ et al., 1997; and of the and of in to from the and and can be localized or and or the Ca2+ the and Table and evidence for the involvement of in Ca2+ at the pollen from the use of the et al., 1995), of through Ca2+ dye to the to pollen for demonstration of so far have been because from the pollen between the membrane and the Ca2+-selective studies also have shown Ca2+ at the root and Felle, et al., 1995). the localized elevation and root growth by that be in maintenance of the et al., that a of the to Ca2+ The root should be more than is the pollen for studies of Ca2+ and signaling. Ca2+ in which the of is in the Ca2+ et al., The Ca2+ in well with the presence of and at the et al., et al., has been shown to to of of the plasma membrane to the cell et al., Whereas Ca2+ imaging that at the of the cell and it not this or during of the A potential with imaging Ca2+ during the early of is the buffering of small localized Ca2+ by the and used an to early Ca2+ in by ratio imaging of with low concentrations of the images of loaded with be of with that the Ca2+ The Ca2+ in root be only during growth and not during root et al., 1997), a role for Ca2+ in growth Although pollen can be by that of Ca2+, root to a in growth direction by of a Ca2+ only transient changes in the direction of which the and underlie the and of the root In to the pollen (Miller et al., to the root et al., However, it is also that root to at relative to the that signal is used to the of the tip with to the root is to and the and in Ca2+ from the cytosol. For is biochemical evidence for a Ca2+ in the plasma membrane (e.g., 1997; et al., 1997), but the has not been even larger in knowledge the of into the of plant The low and size of Ca2+ for from However, for both Ca2+ and that are both and likely from in the The challenge is to understand the of and have the of a detailed into Ca2+ are in of the that is to the biochemical and of and is that be required to an of in and have The is a to in known Ca2+ in This has yielded the of a Ca2+ in higher as a into the et al., The is to transgenic plants with to or block Ca2+ transport pathways. For the has been with a that it et al., 1998). are with to transport pathways in transgenic the associated with calcium with The use of fluorescent and aequorin to changes in [Ca2+]c can only limited on the of Ca2+ signals. localized changes can only from imaging studies or the use of The of as fluorescent et al., 1997), is likely to on Ca2+ signaling. This for studies of and of Ca2+ signals with of signaling pathways. for with