Glutamate-Gated Calcium Fluxes in Arabidopsis 1

It is well accepted that endogenous and environmental signals can influence cellular activities by changing [Ca]cyt (Malhó et al., 1998; Sanders et al., 1999). Despite the importance of this mechanism for coupling stimuli to responses (Malhó et al., 1998), the molecules responsible for generating increases in [Ca]cyt during cell signaling in plants are not known at the genetic level. The results presented here raise the possibility that ligand-gated ion channels in plants such as those predicted by the discovery of ionotropic glutamate receptor (iGluR)like genes in Arabidopsis (Lam et al., 1998) are key components of a Ca influx mechanism important to signal transduction. In animal brains iGluR channels mediate fast chemical transmission across synapses by increasing the permeability of the post-synaptic cell membrane to K, Na, and Ca after binding Glu released by the presynaptic cell (Hille, 1992; Hollmann and Heinemann, 1994; Dingledine et al., 1999). The resulting Ca entry in particular has been associated with long-term potentiation of the synapse, a physiological adaptation important to the learning process (Baudry and Lynch, 1993; Bliss and Collinridge, 1993). The Glu receptor homologs recently identified in plants (Lam et al., 1998) are too divergent from animal iGluRs to know with any certainty what ligand(s) gate them, what ions are conducted in the open state, and in which membrane(s) of the cell they function (Chiu et al., 1999). Thus the identification of iGluR sequences in the Arabidopsis genome raises intriguing questions about the physiological functions of neurotransmitter-gated channels in plant cells. The possibility that Glu gates Ca-permeable channels at the plasma membrane of plant cells was explored by measuring [Ca]cyt in transgenic seedlings expressing aequorin, a Ca-sensitive luminescent protein (Knight et al., 1991). As shown in Figure 1A, Glu application immediately triggered a very large, transient spike in [Ca]cyt. In separate experiments the effect of Glu on membrane potential (Vm) was measured with intracellular microelectrodes inserted into root apices of intact seedlings. Figure 1A also shows that switching the bathing medium from 1 mm KCl to 1 mm K-Glu induced a large and rapid depolarization of the membrane, as would be expected if the abrupt increase in [Ca]cyt was due to Glu opening Ca-permeable channels at the plasma membrane. The average peak change in Vm induced by 1 mm Glu was 55 6 7 mV (n 5 6 seedlings). This positive shift in Vm, though consistent with Glu gating an inward electrogenic Ca current across the plasma membrane, may also be due to secondary effects of the increased [Ca]cyt on other ion transporters. Another scenario to consider is that Glu directly gates channels permeable to ions such as Cl (Cully et al., 1994) in addition to Capermeable channels to cause the depolarization. And last, an electrogenic Glu-uptake mechanism (Boorer et al., 1996) may also contribute to the electrical response. Because these and perhaps other scenarios are not mutually exclusive, more electrophysiological studies of the connection between the large Glu-gated changes in [Ca]cyt and the effect on Vm are warranted. Figure 1B shows that the magnitude and time course of the rapid increase in [Ca]cyt was similar to the well-studied response to cold shock, i.e. in the micromolar concentration range and completed within several seconds (Knight et al., 1991, 1996; Lewis et al., 1997). Treatment with 1 mm Glu induced a response that was typically hundreds of fold higher than the control injection of equimolar KCl, which produced a touch response that may reflect Ca entering the cytoplasm from internal stores (Haley et al., 1995; Legué et al., 1997). The post-peak shoulder apparent in the selected response to Glu was often, but not always observed. Activation of iGluRs by Glu causes very similar Ca changes in cells of the mammalian nervous system (Kirischuk et al., 1999; Obrietan and van den Pol, 1999). If the increase in [Ca]cyt triggered by Glu resulted at least in part from flux across the plasma membrane from the apoplasm, impermeant channel blockers and external chelators of Ca should reduce the response. The results in Figure 2A demonstrate that pretreatment with La, a frequently used blocker of plasma membrane Ca channels, inhibited the Ca spike to the low level induced by the control treatment. Chelating extracellular Ca by pre-treating seedlings with EGTA was similarly inhibitory (Fig. 2B). The combined evidence support our suggestion that Glu triggers an influx of Ca across the plasma membrane and this leads to a dramatic change in [Ca]cyt. Calcium-induced Ca -release from inter1 This work was supported by the National Science Foundation (career award no. IBN–9734478 to E.P.S.). * Corresponding author; e-mail spalding@facstaff.wisc. edu; fax 608 –262–7509.