Plasmalemma Abscisic Acid Perception Leads to RAB18Expression via Phospholipase D Activation in Arabidopsis Suspension Cells

Abstract

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...

Phospholipase D Activation Is an Early Component of the Salicylic Acid Signaling Pathway in Arabidopsis Cell Suspensions

“…This time course of PLD activation is similar to that observed for the response to abscisic acid (Hallouin et al, 2002) in Arabidopsis cells. The PLD activation was dependent on the dose of SA, with a maximum of response obtained with 250 mM SA.…”

Induction of Abscisic Acid-Regulated Gene Expression by Diacylglycerol Pyrophosphate Involves Ca2+ and Anion Currents in Arabidopsis Suspension Cells

“…In Arabidopsis suspension cells, we have demonstrated that ABA provoked RAB18 expression through stimulation of PLD activity (Jeannette et al, 1999;Hallouin et al, 2002). In this study, we show that DGPP is able to trigger the expression of three ABA up-regulated genes (At5g06760, LTI30, and RD29A) in addition to RAB18.…”

Plasma Membrane Depolarization Induced by Abscisic Acid in Arabidopsis Suspension Cells Involves Reduction of Proton Pumping in Addition to Anion Channel Activation, Which Are Both Ca2+ Dependent

“…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;.…”

“…This could suggest a role for the plasma membrane potential in the control of gene expression (Schaller and Frasson, 2001). In previous articles (Ghelis et al, 2000a;Hallouin et al, 2002), we showed that the induction of RAB18 gene expression in response to ABA was dependent on the ABA induced activation of anion channels. Since we now know that plasma membrane depolarization induced by ABA involved both anion channel activation and proton pumping reduction, we have now to determine what signal is responsible for the induction of the RAB18 gene expression: the variation of the anion currents or the plasma membrane depolarization induced by this anion current variation.…”