Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores and [Ca2+]cyt oscillations are a fundamental component of stomatal closure. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a 'calcium clamp' and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term 'calcium-reactive' closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was 'calcium programmed' by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.

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Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt).

Elevations in cytoplasmic calcium ([Ca2 INTRODUCTIONMany physiological stimuli in plant cells induce elevations in cytoplasmic calcium ([Ca 2 ϩ ] cyt ), which is an essential second messenger in plant signal transduction cascades (Bush, 1995; Trewavas and Malhó, 1997). However, thus far, only one report has directly demonstrated a mutation that impairs stimulus-induced [Ca 2 ϩ ] cyt elevations (Bush, 1996). The hormone abscisic acid (ABA) controls a wide variety of stress responses and developmental processes in plants ( Leung and Giraudat, 1998), and [Ca 2 ϩ ] cyt has been proposed to function as a second messenger in several ABA responses (McAinsh et al., 1990;Sheen, 1996Sheen, , 1998 Wu et al., 1997). ABA is produced in response to drought stress and mediates a reduction in stomatal aperture that prevents excessive evaporation-mediated water loss. Stomatal closure is elicited via a reduction in the Cl Ϫ , K ϩ , and organic solute content in the two guard cells that border the stomatal pore (Assmann, 1993; MacRobbie, 1997;Müller-Röber et al., 1998). In guard cells, ABA induces an increase in [Ca 2 ϩ ] cyt , which precedes the reduction in stomatal aperture (McAinsh et al., 1990(McAinsh et al., , 1992Schroeder and Hagiwara, 1990; Gilroy et al., 1991; Irving et al., 1992; Allan et al., 1994; Grabov and Blatt, 1998). The [Ca 2 ϩ ] cyt rise initiates the processes required for guard cell turgor loss through the modulation of ion channels and pumps in both the plasma and vacuolar membranes (Schroeder and Hagiwara, 1989; Hedrich et al., 1990; Fairley-Grenot and Assmann, 1991; Luan et al., 1993; Lemtiri-Chlieh and MacRobbie, 1994; Ward and Schroeder, 1994; Kinoshita et al., 1995; Allen and Sanders, 1996).Studies have indicated that outwardly rectifying K ϩ currents and slow (S-type) anion currents play an important role in the process of ion efflux that drives stomatal closure (reviewed in Assmann, 1993; MacRobbie, 1997;Müller-Röber et al.,1998). Ca 2 ϩ activation of S-type anion currents results in anion efflux and depolarization of the guard cell plasma membrane (Schroeder and Hagiwara, 1989). ABA activates S-type anion currents in guard cells of Arabidopsis (Pei et al., 1997), tobacco (Grabov et al., 1997), and fava bean (Schwarz and Schroeder, 1998). However, ABA activation of anion channels is impaired in guard cells of the Arabidopsis ABA-insensitive mutants abi1 and abi2 (Pei et al., 1997(Pei et al., , 1998. Because the stomata of abi1 and abi2 do not close in 1 To whom correspondence should be addressed. E-mail gallen@ biomail.ucsd.edu; fax 858-534-7108. 2 Current address: Department of Biotechnology, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba 305-8602, Japan. 1786The Plant Cell response to exogenous ABA or drought stress (Roelfsema and Prins, 1995;Pei et al., 1997), these findings emphasize that activation of S-type anion currents is one of the essential early events in the ABA signal transduction cascade in guard cells.The abi1 and abi2 loci encode (semi)dominant mutati...