In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H 1 -ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H 1 pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H 1 -ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca 21 ] cyt increase induced by ABA since increase in [Ca 21 ] cyt enhanced anion channels and impaired H 1 -ATPases.Abscisic acid (ABA) induces the depolarization of the plasma membrane (Thiel et al., 1992). This depolarization has been interpreted as the consequence of the activation of anion channels in stomatal guard cells of Vicia faba (Blatt, 1990;Schroeder and Keller, 1992;Thiel et al., 1992;Ward et al., 1995), Nicotiana benthamiana and Commelina communis (Schwartz et al., 1995;Leonhardt et al., 1999). In addition, we demonstrated that the extracellular perception of ABA in Arabidopsis suspension cells was necessary for the activation of anion channels inducing the plasma membrane depolarization (Ghelis et al., 2000a), and recently we showed that this anion channel stimulation induced by extracellular ABA perception was dependent on phospholipase D activities (Hallouin et al., 2002). In guard cells that are the most studied plant cell model used for the dissection of ABA signaling pathways (Assmann, 1993;Schroeder et al., 2001), two distinct anion channels, rapid anion channels (R-type) and slow anion channels (S-type), were proposed to participate in the plasma membrane depolarization (Schroeder and Keller, 1992;. Both R-type and S-type channels have been suggested to contribute to an initial phase of the depolarization, while maintenance of the depolarized state of the plasma membrane was only attributed to the S-type anion channels (Schroeder and Keller, 1992). The mechanisms by which ABA activates anion channels are not entirely understood . In V. faba guard cells, activation of anion channels by ABA can be observed without variation of the cytoplasm calcium concentration, suggesting that the ABA-induced anion efflux is calcium-independent (Schwarz and Schroeder, 1998). However, numerous data support the calcium dependence of the anion channel activation in response to ABA. Some of the anion channels involved in a long-term plasma membrane depolarization are Ca 21 -sensitive and therefore are activated by an increase in cyto...

SummaryImportant progress has been made regarding the characterization of the ABA signalling components using genetic and molecular approaches (Leung and Giraudat, 1998). However, we do not yet know the mechanism of ABA perception. Conflicting results concerning the site of ABA perception have been published. The prevailing view is that since ABA controls many responses, different sites of perception for ABA might exist. In order to establish the cellular localisation of the ABA receptors in Arabidopsis thaliana suspension cells, we developed two physiological tests based upon the capacity of impermeant ABA-BSA conjugate to mimic permeant free ABA effects. We show that purified ABA-BSA conjugate is able to trigger RAB18 gene expression and that this response is strictly due to the natural (⍣)-ABA enantiomer. The rate of RAB18 gene expression was independent of the level of ABA uptake by the cells. Using the voltage-clamp technique we show that ABA-BSA, similarly to ABA, evokes a membrane depolarization and activates time-and voltage-dependent outward rectifying currents (ORC). We demonstrate that these ORC are due to a K ⍣ efflux as assessed by tail currents and specific inhibition by both tetraethylammonium (TEA) and Ba 2⍣ . These observations provide evidence in favour of an extracellular site for ABA perception.

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K ϩ channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca 2ϩ influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.Abscisic acid (ABA) regulates seed maturation and germination, adaptation of plants to water shortage, cold, and high salinity (Leung and Giraudat, 1998). Several ABA transduction mutants have been isolated in Arabidopsis in which diverse loci affecting ABA response have been identified (for review, see Merlot and Giraudat, 1997;Leung and Giraudat, 1998). Among the best characterized are the ABAinsensitive abi-1 and abi-2 mutations, which affect protein phosphatases (Leung et al., 1997); the abi-3 (Giraudat et al., 1992;Parcy et al., 1994), abi-4 (Finkelstein et al., 1998), and abi-5 (Finkelstein and Lynch, 2000), which are mutated in transcription processes; and the ABA-hypersensitive era1 mutant, deleted for the ␤-subunit of a farnesyl transferase which acts as a negative regulator (Cutler et al., 1996). In guard cells, ABA transduction pathways have been extensively analyzed. Stomatal aperture is controlled by ABA through the activation of anion currents, which depolarizes the plasma membrane and promotes the activation of outward-rectifying K ϩ currents (Schroeder et al., 2001). In aleurone cells, ABA causes a decrease in cytoplasmic Ca 2ϩ content, which is necessary for the inhibition of GA promotion of ␣-amylase activity (Ritchie and Gilroy, 1998). In Arabidopsis suspension cells, similar ion channel activation has been observed (Jeannette et al., 1999; Ghelis et al., 2000a Ghelis et al., , 2000bTakahashi et al., 2001). However, the precise sequence of events triggered by ABA in these models remains unknown.Recent research has provided new data about the role of phospholipases...