Research ArticleCELL BIOLOGY AND SIGNAL TRANSDUCTION

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

Christine Zalejski, Sophie Paradis, Régis Maldiney, Yvette Habricot, Emile Miginiac, Jean-Pierre Rona, Emmanuelle Jeannette

Published August 2006. DOI: https://doi.org/10.1104/pp.106.080218

Abstract

Diacylglycerol pyrophosphate (DGPP) was recently shown to be a possible intermediate in abscisic acid (ABA) signaling. In this study, reverse transcription-PCR of ABA up-regulated genes was used to evaluate the ability of DGPP to trigger gene expression in Arabidopsis (Arabidopsis thaliana) suspension cells. At5g06760, LTI30, RD29A, and RAB18 were stimulated by ABA and also specifically expressed in DGPP-treated cells. Use of the Ca2+ channel blockers fluspirilene and pimozide and the Ca2+ chelator EGTA showed that Ca2+ was required for ABA induction of DGPP formation. In addition, Ca2+ participated in DGPP induction of gene expression via stimulation of anion currents. Hence, a sequence of Ca2+, DGPP, and anion currents, constituting a core of early ABA-signaling events necessary for gene expression, is proposed.

Plants use various strategies to endure water shortage. In vegetative tissues, rapid limitation of water loss occurs through the closure of stomata, whereas long-term adaptation to drought and seed development are accompanied by the expression of a set of genes encoding proteins involved in protective processes. Abscisic acid (ABA) accumulates under hydric stress and is essential, via the control of guard cell movements and gene expression, for the establishment of drought resistance processes. Regulation of many stress-induced genes is mediated through ABA; however, the existence of ABA-independent pathways is also established (Ishitani et al., 1997; Shinozaki and Yamaguchi-Shinozaki, 2000; Seki et al., 2003; Riera et al., 2005). Among the set of ABA-inducible genes, late-embryogenesis abundant (LEA) protein genes are expressed during seed maturation and in vegetative parts of plants exposed to water limitation (Skriver and Mundy, 1990; Roberts et al., 1993; Bray, 1997). The signaling cascade leading to ABA-regulated genes is currently well understood with respect to DNA transcription. Promoters of ABA-responsive genes have specific ABA cis-acting elements able to bind trans-acting factors (Finkelstein et al., 2002). By contrast, knowledge of the sequence of events linking ABA perception to nuclear events is more fragmentary, although an ABA receptor was recently identified (Razem et al., 2006). Several kinases and phosphatases, G proteins, ion channels, Ca2+, cADP-Rib, and lipid compounds are known to interact in complex signaling networks, but targets and partners of these elements are mostly not identified (Leung and Giraudat, 1998; Finkelstein and Rock, 2002).

Among the second messengers of ABA, Ca2+ plays an important role through changes in cytosolic Ca2+ concentration. For example, in GA-treated aleurone protoplasts, ABA diminishes cytosolic Ca2+ concentration (Ritchie and Gilroy, 1998). In guard cells, ABA provokes a fast rise in cytosolic Ca2+ concentration (McAinsh et al., 1990) that regulates anion efflux (Schroeder and Hagiwara, 1989; Hedrich et al., 1990), inhibits inward K+ currents (Schroeder and Hagiwara, 1989; Grabov and Blatt, 1999), and triggers vacuolar K+ release (MacRobbie, 2000).

Lipids and phospholipases are involved in most of the signaling pathways in plants (Meijer and Munnik, 2003; Testerink and Munnik, 2005). In ABA-regulated processes, phosphatidic acid (PA) produced by phospholipase D (PLD) activity and/or by phospholipase C coupled to diacylglycerol kinase is a messenger in guard cell movement, leaf senescence, and inhibition of α-amylase production in aleurone protoplasts (Fan et al., 1997; Ritchie and Gilroy, 1998; Jacob et al., 1999). Moreover, the importance of PA in ABA responses was recently underlined by the demonstration that PA produced by PLDα was bound to the protein phosphatase 2C ABI1 to regulate its activity (Zhang et al., 2004).

In plants and yeast (Saccharomyces cerevisiae), PA can specifically be phosphorylated by PA kinase activity to form diacylglycerol pyrophosphate (DGPP; Wissing and Behrbohm, 1993a; Munnik et al., 1996; Wu et al., 1996). DGPP is a minor phospholipid, which, concomitantly to PA, increases in response to various abiotic and biotic stresses (Pical et al., 1999; Munnik et al., 2000; van der Luit et al., 2000; den Hartog et al., 2001; de Jong et al., 2004). After ABA application, the level of DGPP was also increased in Arabidopsis (Arabidopsis thaliana) suspension cells and seeds (Katagiri et al., 2005; Zalejski et al., 2005). Consequently, DGPP was hypothesized to play a role in plant signaling and, especially, in ABA responses (van Schooten et al., 2006). Regulation of the metabolic pathway that leads to ABA-induced DGPP accumulation is beginning to be elucidated. The lipid phosphate phosphatases (LPPs) responsible for the dephosphorylation of DGPP to PA and of PA to diacylgylcerol are encoded by four genes in Arabidopsis (Pierrugues et al., 2001; Katagiri et al., 2005). Expression of AtLPP1 was shown to be induced by genotoxic stress and to be present in leaves (Pierrugues et al., 2001), whereas AtLPP2 and AtLPP3 were found in seeds where AtLPP2 is a negative regulator of ABA signaling during germination (Katagiri et al., 2005). PA kinase activity was found to be associated with plasma membranes in various plant species but, unfortunately, a PA kinase gene has not yet been identified (Wissing and Behrbohm, 1993b).

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. Experiments performed to locate DGPP versus Ca2+ influx demonstrated that Ca2+ occurs both up- and downstream of DGPP formation in the ABA-signaling cascade that leads to gene expression. Our observations support the existence of an early core of common events necessary for ABA-induced expression of several genes.

RESULTS

DGPP Induces Expression of ABA Up-Regulated Genes

In Arabidopsis suspension cells, we previously observed that DGPP content was increased consecutively to ABA treatment and that the application of dioleoylDGPP was able to trigger the expression of RAB18 (Zalejski et al., 2005). To evaluate the importance of DGPP for the expression of genes induced by ABA, three supplementary genes were chosen. At5g06760, LTI30 (also named DHNXERO2, At3g50970), and RD29A (also named LTI78 or COR78, At5g52310) were selected because they were characterized as ABA up-regulated genes (Leonhardt et al., 2004). Reverse transcription (RT)-PCR analysis of At5g06760, LTI30, and RD29A shows that their expression was stimulated by 10 μm ABA in the suspension cells within 3 h (Fig. 1 ; see also Supplemental Fig. 1). Application of dioleoylDGPP (300 μm, 3 h) also induced expression of these genes (Fig. 1). Expression of At5g06760 and LTI30 was neither induced by dioctanoylDGPP nor by dioctanoylPA and dioleoylPA. Weak expression of RD29A was recorded with the short fatty acid chains dioctanoylDGPP and dioctanoylPA, whereas dioleoylPA was less efficient than dioleoylDGPP in triggering RD29A expression (Fig. 1). From these data, we conclude that dioleoylDGPP was the most efficient phospholipid able to induce expression of several ABA up-regulated genes. The efficiency of dioleoylDGPP may be attributed to the long fatty acid unsaturated chains associated with the pyrophosphate moiety of the molecule.

Figure 1.
Figure 1.

DioleoylDGPP induces expression of ABA up-regulated genes. RT-PCR analysis of At5g06760, LTI30, and RD29A expression in Arabidopsis suspension cells is shown. Total RNA (2.5 μg) was isolated from cells incubated for 3 h with 10 μm ABA or 300 μm dioctanoylPA, dioleoylPA, dioctanoylDGPP, or dioleoylDGPP. PCR products were separated on 2% agarose gel and stained with ethidium bromide. ACT2 was amplified as a control. The data presented are representative of three independent experiments.

Role of Ca2+ in DGPP Stimulation of Gene Expression

In Arabidopsis suspension cells, we observed that ABA caused a Ca2+ influx via fluspirilene- and pimozide-sensitive channels (Ghelis et al., 2000b). This Ca2+ entrance was required for RAB18 expression. Hence, we questioned whether Ca2+ was also necessary for ABA- and DGPP-induced At5g06760, LTI30, and RD29A expression. Application of fluspirilene (50 μm) or pimozide (50 μm) decreased expression of At5g06760, LTI30, and RD29A triggered by ABA (Fig. 2 ). Thus, it appears that the increase in cellular Ca2+ concentration was, as well as DGPP, involved in ABA regulation of At5g06760, LTI30, and RD29A expression. To determine whether DGPP and Ca2+ acted independently or belonged to the same ABA-signaling cascade, two experiments were performed. First, we measured the DGPP level when ABA-treated cells were supplemented or not with Ca2+ channel inhibitors or a Ca2+ chelator. Cells were labeled with 33P for 18 h and incubated for 15 min with fluspirilene (50 μm), pimozide (50 μm), or the Ca2+ chelator EGTA (5 mm), and then with or without ABA (10 μm) for 20 min. Lipids were then extracted, DGPP was separated by a two-dimensional (2-D) thin-layer chromatography (TLC) system and quantified. In ABA-treated cells, the amount of DGPP was increased 7-fold (Fig. 3 ). Fluspirilene and pimozide alone had no effect on DGPP basal level but, when they were added simultaneously with ABA, accumulation of DGPP was inhibited. Addition of fluspirilene inhibited DGPP formation only partially, whereas pimozide caused total inhibition of ABA-induced DGPP accumulation. EGTA alone triggered a slight increase in DGPP level and when added to ABA, provoked weak inhibition of DGPP formation (Fig. 3). Second, expression of At5g06760, LTI30, RD29A, and RAB18 was recorded in cells simultaneously treated with DGPP and the Ca2+ channel inhibitors. Expression of the four genes induced by DGPP was diminished in the presence of fluspirilene and pimozide. Furthermore, pimozide was the most efficient inhibitor of DGPP action on gene expression (Fig. 4 ).

Figure 2.
Figure 2.

ABA regulation of gene expression requires Ca2+. RT-PCR analysis of At5g06760, LTI30, RD29A, and RAB18 expression in Arabidopsis suspension cells is shown. Total RNA (2.5 μg) was isolated from control cells (C) or cells incubated for 3 h with fluspirilene (F; 50 μm) or pimozide (P; 50 μm) and without or with ABA (10 μm). PCR products were separated on 2% agarose gel and stained with ethidium bromide. ACT2 was amplified as a control. The data presented are representative of three independent experiments.

Figure 3.
Figure 3.

ABA stimulation of DGPP formation is impaired when Ca2+ influx is blocked. Cells were labeled for 18 h with Embedded Image, incubated for 15 min with fluspirilene (F; 50 μm), pimozide (P; 50 μm), or EGTA (5 mm), and then for 20 min with ABA (10 μm) before lipid extraction. DGPP was separated with a 2-D TLC system and quantified. The relative DGPP level is expressed with reference to the level in the control (untreated cells). The results show the mean ± sem; n = 3.

Figure 4.
Figure 4.

DGPP acts upstream of Ca2+ to trigger the expression of ABA up-regulated genes. RT-PCR analysis of At5g06760, LTI30, RD29A, and RAB18 expression in Arabidopsis suspension cells is shown. Total RNA (2.5 μg) was isolated from cells incubated for 3 h with 300 μm dioleoylDGPP and fluspirilene (F; 50 μm) or pimozide (P; 50 μm). PCR products were separated on 2% agarose gel and stained with ethidium bromide. ACT2 was amplified as a control. For gene expression in the control, F-, or P-treated cells, see Figure 2. The data presented are representative of three independent experiments.

Taken together, data from Figures 2 to 4 demonstrate that Ca2+ acts both up- and downstream of DGPP formation in the ABA pathway, leading to At5g06760, LTI30, RD29A, and RAB18 expression.

DGPP Induces Gene Expression through Ca2+ Stimulation of Anion Currents

To further explore how DGPP and Ca2+ act in the ABA-signaling pathway that leads to gene expression, we studied plasmalemma anion currents because they were shown to be stimulated by DGPP and necessary for RAB18 expression (Ghelis et al., 2000a; Brault et al., 2004; Zalejski et al., 2005). We first examined the effect of the anion channel blocker 9-anthracene carboxylic acid (9-AC) on gene expression. Figure 5 shows that 9-AC (50 μm) partially inhibited ABA-induced expression of At5g06760, whereas expression of LTI30, RD29A, and RAB18 was totally inhibited. When gene expression was triggered by DGPP, addition of 9-AC also inhibited gene expression. Therefore, DGPP action on gene expression required anion channel activity.

Figure 5.
Figure 5.

DGPP stimulation of gene expression occurs through anion current activation. RT-PCR analysis of At5g06760, LTI30, RD29A, and RAB18 expression in Arabidopsis suspension cells is shown. Total RNA (2.5 μg) was isolated from cells incubated with dioleoylDGPP (300 μm), 9-AC (50 μm), and/or ABA (10 μm) for 3 h. PCR products were separated on 2% agarose gel and stained with ethidium bromide. ACT2 was amplified as a control. The data presented are representative of three independent experiments.

Whole-cell voltage-clamp assays were performed to elucidate the relation between Ca2+ and plasma membrane anion currents. DioleoylDGPP stimulated plasma membrane anion currents that induced membrane depolarization (Fig. 6, A, B, and D ). DGPP evoked 300% activation in anion current intensity at −200 mV compared to the control (Fig. 6F). Addition of 9-AC to DGPP-treated cells reversed DGPP action (Fig. 6, A and F). When fluspirilene or pimozide were added after DGPP, the increase in anion currents was reversed (from 300% to 200% with fluspirilene and from 300% to 100% with pimozide) and repolarization of the plasma membrane was observed (Fig. 6, B, D, and F). Conversely, when fluspirilene or pimozide were added prior to DGPP, no significant increase in anion currents was recorded and no depolarization of the plasma membrane was observed (Fig. 6, C, E, and F). These data demonstrate that DGPP stimulation of plasmalemma anion currents is mediated by the activity of Ca2+ channels. Moreover, this step is necessary for expression of several ABA-regulated genes.

Figure 6.
Figure 6.

DGPP activation of anion currents requires Ca2+ channel activity. Whole-cell currents were activated by a depolarizing prepulse (+100 mV for 3.5 s; data not shown). Then, hyperpolarizing pulses ranging from −200 to +40 mV, in 40-mV steps, were applied for 6.5 s. Representative current traces at −200 mV and the current-voltage relationships determined for currents recorded after 10-s deactivation are shown. Holding potential was −40 mV. Currents were recorded from intact cells before (control) and 1 min after application of dioleoylDGPP (DGPP) or inhibitors. A, 100 μm DGPP for 5 min, then 100 μm anthracene-9-carboxylic acid (9-AC). B, 100 μm DGPP for 5 min, then 50 μm fluspirilene (DGPP + F). C, 50 μm fluspirilene (F) for 5 min, then 100 μm DGPP (F + DGPP). D, 100 μm DGPP for 5 min, then 50 μm pimozide (DGPP + P). E, 50 μm pimozide (P) for 5 min, then 100 μm DGPP (P + DGPP). F, Relative anion current intensities recorded at −200 mV, in percentage. Data represent the means of three replications ± sd.

DISCUSSION

In this study, we demonstrated that expression of the ABA-regulated genes At5g06760, LTI30, and RD29A can be triggered by dioleoylDGPP in Arabidopsis cells. This observation extends findings from our previous study that was restricted to the role of dioleoylDGPP on RAB18 expression (Zalejski et al., 2005). Because the DGPP level was transiently increased after ABA perception (Zalejski et al., 2005), our results provided further evidence for the role of DGPP as a plant second messenger of ABA. By comparison with dioleoylDGPP, the nonphysiological dioctanoylDGPP was less efficient in triggering RD29A expression and unable to trigger At5g06760 and LTI30 expression. Thus, the effect recorded with dioctanoylDGPP could be a pharmacological-like effect. Interestingly, our results also showed that RD29A was expressed under dioleoylPA treatment. However, the effect of dioleoylPA was not recorded with all three of the chosen genes; for this reason, we assumed that PA's ability to mimic ABA was due to its own specificity and not to its subsequent phosphorylation into DGPP. This hypothesis is in accordance with the observation that PA production played a key role in the induction of RD29A in cold-treated Arabidopsis suspension cells (Vergnolle et al., 2005).

At5g06760 encodes a LEA protein of group 1 and LTI30 and RAB18 encode LEA proteins of group 2, also called dehydrins (Ramanjulu and Bartels, 2002). Due to their high hydrophilicity, LEA proteins are thought to play a major role in plant, animal, and prokaryote resistance to water deficit (Garay-Arroyo et al., 2000; Browne et al., 2002; Wise and Tunnacliffe, 2004). In plants, the predicted protective function of LEA proteins is further reinforced by many studies that correlate plant tolerance against drought, low temperature, and salinity with LEA protein expression (Lang and Palva, 1992; Lang et al., 1994; Welin et al., 1994; Nylander et al., 2001; Ramanjulu and Bartels, 2002). Moreover, in yeast, introduction of plant LEA confers increased resistance to freezing (Imai, 1996) and osmotic stress (Swire-Clark and Marcotte, 1999). Accordingly, increases in DGPP observed under osmotic stress (Pical et al., 1999; Munnik et al., 2000), salinity (Pical et al., 1999), or biotic stresses (van der Luit et al., 2000; den Hartog et al., 2001; de Jong et al., 2004) may induce LEA expression in vegetative tissues and therefore may be essential for plant survival in stress conditions. In ABA-treated seeds, the increase observed in DGPP level (Katagiri et al., 2005) could also regulate LEA expression and therefore participate in seed resistance against desiccation.

In ABA-treated cells, when Ca2+ influx was blocked or Ca2+ was chelated in the cell culture medium, the increase in DGPP level was impaired (Fig. 3). This observation demonstrates that the increase in DGPP was dependent on Ca2+ entry through diphenylbutylpiperidine-sensitive channels. Thus, the question is to determine how Ca2+ could participate in the control of the DGPP level. DGPP is the phosphorylated form of PA produced by PA kinase activity (Wissing and Behrbohm, 1993a, 1993b; Munnik et al., 1996). Conversely, DGPP can be dephosphorylated by the DGPP phosphatase activity of LPP (Pierrugues et al., 2001; Katagiri et al., 2005). Thus, the level of DGPP depends upon these two enzymatic activities and Ca2+ may either activate PA kinase or inhibit LPP or regulate both enzyme activities. Unfortunately, control of DGPP metabolism is currently not fully understood and a PA kinase gene has not been identified so far in plants or yeast (van Schooten et al., 2006). Nevertheless, PA kinase activity has been measured in several plant species (Wissing and Behrbohm, 1993b; Wissing et al., 1994). Accordingly, DGPP was also found in many species like Chlamydomonas (Munnik et al., 1996, 2000), Craterostigma plantagineum (Munnik et al., 2000), and tomato (Lycopersicon esculentum; van der Luit et al., 2000). However, to our knowledge, Ca2+ has never been reported as required for PA kinase regulation. Besides that, recent studies have given valuable information on plant LPPs that have added to data on yeast DGPP phosphatases (Oshiro et al., 2003). In Arabidopsis, AtLPP1 was isolated by differential displays in UV-radiated cells and three more genes (AtLPP2, AtLPP3, and AtLPP4) were found, based on genomic sequence similarities (Pierrugues et al., 2001; Katagiri et al., 2005). The predicted structure of the four plant LPPs is closely related to yeast LPP with six transmembrane-spanning domains and three domains involved in phosphatase activity (Oshiro et al., 2003). In yeast, inhibition of DGPP phosphatase activity by Ca2+ was reported (Wu et al., 1996). Therefore, it is possible that in Arabidopsis suspension cells, Ca2+ inhibited DGPP phosphatase activity that participates in the regulation of the DGPP level. In addition, it is likely that Ca2+ regulates a signaling intermediate upstream of PA kinase or DGPP phosphatase activities. The existence of a Ca2+-binding domain on PLDs (Wang, 2001) supports the hypothesis that Ca2+ may also regulate the DGPP level through PLD-mediated PA synthesis. Finally, posttranslational modifications of the enzyme responsible for DGPP metabolism may also occur.

Electrophysiological experiments have shown that dioleoylDGPP enhanced the activity of 9-AC-sensitive anion channels. Furthermore, RT-PCR analysis showed that dioleoylDGPP stimulation of gene expression occurred via anion current activation. Anion currents are involved in many physiological processes, including plant responses to pathogens (Wendehenne et al., 2002) or oxidative stress (Cazalé et al., 1998), but their precise role in signaling is poorly understood (Barbier-Brygoo et al., 2000). In suspension cells, the mechanism by which outward anion currents are required for gene transcription is unclear. One possibility is that plasmalemma depolarization induced by anion efflux (Brault et al., 2004) could be responsible for the activation of unknown targets. Otherwise, efflux of certain anions could be part of this specific response. In guard cells, ABA-regulated anion efflux plays a key osmotic role because it participates in loss of turgor (Blatt, 2000). In this study, we observed that addition of Ca2+ channel inhibitors impaired the activity of anion currents triggered by dioleoylDGPP. Hence, in accordance with several previous observations (Ghelis et al., 2000a, 2000b; Brault et al., 2004), a link between Ca2+ and anion channels is suggested in Arabidopsis suspension cells. In guard cells, the dependence of anion channel activities on the cytosolic Ca2+ concentration increase was demonstrated (Schroeder and Hagiwara, 1989; Hedrich et al., 1990; Blatt, 2000), although activation of anion channels may also occur independently of Ca2+ (Schwarz and Schroeder, 1998; Levchenko et al., 2005). Thus, ABA regulation of anion currents in suspension cells is similar to that in guard cells.

Altogether, our results support the existence of an early signaling plasma membrane core of events necessary for ABA induction of gene expression. The arguments in favor of plasmalemma localization of DGPP, Ca2+ channels, and anion channels can be summarized as follows. First, PA kinase activity was associated with plasmalemma in various species, thus strongly suggesting that metabolism of DGPP occurs in the plasma membrane (Wissing and Behrbohm, 1993a). Moreover, in Arabidopsis suspension cells, mass spectrometry identification of AtLPP3 in the plasmalemma fraction (Marmagne et al., 2004) is consistent with the prediction of transmembrane domains in plant LPP (Pierrugues et al., 2001) and the membrane location of yeast (Han et al., 2001) and Arabidopsis LPP (Katagiri et al., 2005). Although we have no information on DGPP cellular transport, data from suspension cells are in favor of early intervention of DGPP in plasmalemma, where ABA perception occurs (Jeannette et al., 1999). Second, the cellular pool of Ca2+ involved in DGPP formation and the one involved in stimulation of anion currents originate, at least partially, from extracellular Ca2+. Indeed, application of pimozide, fluspirilene, or EGTA impaired both the decrease in extracellular Ca2+ (Ghelis et al., 2000b) and, conversely, the increase in cytosolic Ca2+ triggered by ABA (Brault et al., 2004). Third, DGPP application enhances plasmalemma anion currents. This working model is obviously incomplete, but it provides a framework for the search for missing links, especially the DGPP targets.

Interestingly, a transcriptomics approach that was developed in guard cell protoplasts showed that At5g06760, LTI30, and RAB18 genes were ABA induced in guard cells (Leonhardt et al., 2004). In addition, immunolocalization assays have shown that RAB18 was located in guard cells (Nylander et al., 2001). Albeit for one given gene the signaling pathway leading to its expression is likely dependent on the cell type, DGPP could play a role in ABA signaling leading to LEA expression in guard cells. The presence of DGPP has not, so far, been demonstrated in guard cells, but it was reported that PA could mimic ABA in stomatal closure (Jacob et al., 1999). Furthermore, the ABA-signaling components present in suspension cells (i.e. Ca2+ and anion currents) also play key roles in guard cells. Consequently, it is possible that, after ABA perception, a common module of plasmalemma components triggers both guard cell shrinkage and LEA gene expression. Hence, a specific role of DGPP could be to relay ABA in the induction of gene expression. Phosphorylation of PA in DGPP would be involved in several cellular responses.

MATERIALS AND METHODS

Arabidopsis Suspension Cells

Arabidopsis (Arabidopsis thaliana) ecotype Columbia cells were obtained by Axelos et al. (1992) and cultured at 24°C, under continuous white light (40 μE m−2 s−1) with an orbital agitation at 130 rpm in 500-mL Erlenmeyer flasks containing 200 mL of culture medium (Jouanneau and Péaud-Lenoël, 1967). A 25-mL aliquot of suspension cells was transferred to fresh medium every week. All experiments were conducted on 3-d-old cells. The pH of the culture medium was 6.8 and viability of cells during the experimental treatment was systematically checked with trypan blue tests (data not shown).

Chemicals

ABA, pimozide, fluspirilene, EGTA, 9-AC, dioleoylPA, and dioctanoylPA were from Sigma. DioleoylDGPP and dioctanoylDGPP were from Avanti Polar Lipids. ABA, pimozide, and fluspirilene were added in dimethylsulfoxide at 0.1% final concentration in suspension cells. EGTA was added in water and 9-AC was diluted in methanol (0.1% final concentration). Lipids were sonicated in 1 mL of culture medium, four times, for 1 min at 4°C, and then added to suspension cells.

Lipid Labeling and Measurement of DGPP Level

Measurement of DGPP content was performed as previously described (Zalejski et al., 2005). Cells were labeled with Math (74 Bq/mL of suspension) for 18 h. Lipids from 2.5 g of vacuum-filtrated cells were first separated on K+ oxalate-activated plates with an alkaline solvent chloroform:methanol:ammonia:water (90:70:4:16; v/v/v/v) as described by Munnik et al. (1995). Then the silica strip containing DGPP boarded down with phosphatidylinositol phosphate and up with PA was scrapped from TLC plates and extracted in methanol. DGPP was separated in a 2-D system described by Lepage (1967): first migration in chloroform:methanol:water (130:50:8; v/v/v), second migration in chloroform:acetone:methanol:acetic acid:water (100:40:20:20:10; v/v/v/v/v). Radiolabeled lipid spots were visualized with PhosphorImager (Storm; Molecular Dynamics) and analyzed with ImageQuant software (Amersham). Endogenous DGPP was identified by reference to the migration of standard DGPP. The relative DGPP level was expressed with reference to the level in untreated cells.

RT-PCR Analyses

Five milliliters of suspension cells were treated with ABA, lipids, and the above-mentioned chemicals for 3 h under the culture conditions. Then, cells were collected by vacuum filtration and total RNA was extracted with LiCl according to the protocol previously described (Verwoerd et al., 1989). For RT-PCR, 2.5 μg of total RNA were treated with 1 μL of DNase I (1 unit/μL; Sigma) added to 1 μL of DNase I buffer in a final volume of 8 μL. After 25 min at room temperature, 1 μL of Stop solution (Sigma-Aldrich) was added. The RT mix was prepared with 1 μL of 70 μm oligo(dT), 1 μL of 10 mm dNTP mix (Finnzymes), 1 μL of Moloney murine leukemia virus RTase (200 units/μL; Finnzymes), 1 μL of Out Ribonuclease Inhibitor (40 units/μL; Invitrogen), and 2 μL of reaction buffer (10×; Finnzymes) in a final volume of 9 μL. This RT mix was added to the RNA treated by Dnase I (final volume 20 μL) and cDNA synthesis was performed at 42°C for 60 min. The RTase was then inactivated at 70°C for 10 min. Two microliters of synthesized cDNA were used for PCR amplification by 0.5 units of Taq DNA polymerase (New England Biolabs) in a total volume of 25 μL under the following conditions: 94°C for 2 min, followed by 27 cycles consisting of 15 s at 94°C then 30 s at 59°C for At5g06760, LTI30, RAB18, or 64°C for RD29A and 1 min at 72°C. The procedure was ended at 72°C for 5 min. PCR was performed for 27 cycles within a linear range of amplification of At5g06760, LTI30, RD29A, RAB18, and ACT2 genes. The number and temperature of cycles were optimized for each specific primer pair. We used the following oligonucleotide primers (Eurogentec): At5g06760, forward 5′-GAGGAAAGTGTACGGTTTG-3′ and reverse 5′-CGTCATATCGCTCGCC-3′; LTI30, forward 5′-AAGATTAAAGAGCAACTGCC-3′ and reverse 5′-AACGAAACCAGAAGTAGATATT-3′; RD29A, forward 5′-CGATGCACCAGGCGTAACAGG-3′ and reverse 5′-CCAGCTCAGCTCCTGACTCGTC-3′; RAB18, forward 5′-TTGGGAGGAATGCTTCACC-3′ and reverse 5′-TTGTTCGAAGCTTAACGGC-3′; and ACT2, forward 5′-AACATTGTGCTCAGTGGTGG-3′ and reverse 5′-TCATCATACTCGGCCTTGG-3′. Nine microliters of the PCR products were loaded and separated on 2% agarose Tris-acetate EDTA gel and visualized by ethidium bromide (2 mg/L) for photography.

Electrophysiology

Voltage-clamp measurements of whole-cell currents from intact cells were carried out at room temperature (20°C–22°C) using the technique of the discontinuous single-voltage-clamp microelectrode (Ghelis et al., 2000a). Cells were immobilized by means of a microfunnel (approximately 30–80 μm o.d.) and controlled by a pneumatic micromanipulator (de Fonbrune). Impalements were carried out with borosilicate capillary glass microelectrodes (0.5-μm diameter) filled with 600 mm KCl (electrical resistance from 50–100 MΩ). In this technique, both current passing and voltage recording used the same microelectrode. Sonicated dioleoylDGPP, 9-AC, pimozide, and fluspirilene were diluted in the bathing medium. The membrane potential was held at −40 mV. Anion currents were activated by a depolarizing prepulse (+100 mV for 3.5 s), then hyperpolarizing pulses ranging from −200 to +40 mV in 40-mV steps for 6.5 s were imposed. We systematically checked that cells were correctly clamped by comparing the protocol voltage values with those really imposed.

Acknowledgments

We thank Dr. M. Brault for cell cultures and assistance with electrophysiological experiments. We acknowledge Dr. B. Sotta and A. Zachowski for critical reading of the manuscript and helpful discussions.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Emmanuelle Jeannette (ema{at}ccr.jussieu.fr).

  • Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.080218.

  • ↵1 This work was supported in part by the Ministère de l'Education Nationale, France (grants to C.Z. and S.P.).

  • ↵2 These authors contributed equally to the paper.

  • ↵[W] The online version of this article contains Web-only data.

  • Received March 16, 2006.
  • Revised May 2, 2006.
  • Accepted May 23, 2006.
  • Published June 9, 2006.

LITERATURE CITED

  1. ↵
    Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol Biochem 30: 123–128
    OpenUrl
  2. ↵
    Barbier-Brygoo H, Vinauger M, Colcombet J, Ephritikhine G, Frachisse J-M, Maurel C (2000) Anion channels in higher plants: functional characterization, molecular structure and physiological role. Biochim Biophys Acta 1465: 199–218
    OpenUrlPubMed
  3. ↵
    Blatt M (2000) Cellular signaling and volume control in stomatal movements in plants. Annu Rev Cell Dev Biol 16: 221–241
    OpenUrlCrossRefPubMed
  4. ↵
    Brault M, Amiar Z, Pennarun A-M, Monestiez M, Zhang Z, Cornel D, Dellis O, Knight H, Bouteau F, Rona J-P (2004) 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. Plant Physiol 135: 231–243
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Bray E (1997) Plant responses to water deficit. Trends Plant Sci 2: 48–54
    OpenUrlCrossRef
  6. ↵
    Browne J, Tunnacliffe A, Burnell A (2002) Plant desiccation gene found in a nematode. Nature 416: 38
    OpenUrlCrossRefPubMed
  7. ↵
    Cazalé A-C, Rouet-Mayer M-A, Barbier-Brygoo H, Mathieu Y, Laurière C (1998) Oxidative burst and hypoosmotic stress in tobacco cell suspensions. Plant Physiol 116: 659–669
    OpenUrlAbstract/FREE Full Text
  8. ↵
    de Jong C, Laxalt A, Bargmann B, de Wit P, Joosten M, Munnik T (2004) Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction. Plant J 39: 1–12
    OpenUrlCrossRefPubMed
  9. ↵
    den Hartog M, Musgrave A, Munnik T (2001) Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation. Plant J 25: 55–60
    OpenUrlCrossRefPubMed
  10. ↵
    Fan L, Zheng S, Wang X (1997) Antisense suppression of phospholipase Dα retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell 9: 2183–2196
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Finkelstein R, Gampala S, Rock C (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell 14: S15–S45
    OpenUrlFREE Full Text
  12. ↵
    Finkelstein R, Rock C (2002) Abscisic acid biosynthesis and response. In EM Meyerowitz, CR Somerville, eds, Arabidopsis, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 52
  13. ↵
    Garay-Arroyo A, Colmenero-Flores J, Garciarrubio A, Covarrubias A (2000) Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. J Biol Chem 275: 5668–5674
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Ghelis T, Dellis O, Jeannette E, Bardat F, Cornel D, Miginiac E, Rona J-P, Sotta B (2000a) Abscisic acid specific expression of RAB18 involves activation of anion channels in Arabidopsis thaliana suspension cells. FEBS Lett 474: 43–47
    OpenUrlCrossRefPubMed
  15. ↵
    Ghelis T, Dellis O, Jeannette E, Bardat F, Miginiac E, Sotta B (2000b) Abscisic acid plasmalemma perception triggers a Ca2+ influx essential for RAB18 gene expression in Arabidopsis thaliana suspension cells. FEBS Lett 483: 67–70
    OpenUrlCrossRefPubMed
  16. ↵
    Grabov A, Blatt MR (1999) A steep dependence of inward-rectifying potassium channels on cytosolic free calcium concentration increase evoked by hyperpolarization in guard cells. Plant Physiol 119: 277–288
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Hallouin M, Ghelis T, Brault M, Bardat F, Cornel D, Miginiac E, Rona J-P, Sotta B, Jeannette E (2002) Plasmalemma ABA perception leads to RAB18 expression via phospholipase D activation in Arabidopsis thaliana suspension cells. Plant Physiol 130: 265–272
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Han G, Johnston C, Chen X, Athensteadt K, Daum G, Carman G (2001) Regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase by zinc. J Biol Chem 276: 10126–10133
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Hedrich R, Busch H, Raschke K (1990) Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells. EMBO J 9: 3889–3892
    OpenUrlPubMed
  20. ↵
    Imai R (1996) A LEA class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae. Gene 170: 243–248
    OpenUrlCrossRefPubMed
  21. ↵
    Ishitani M, Xiong L, Stevenson B, Zhu J-K (1997) Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell 9: 1935–1949
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Jacob T, Ritchie S, Assmann SM, Gilroy S (1999) Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity. Proc Natl Acad Sci USA 96: 12192–12197
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Jeannette E, Rona J-P, Bardat F, Cornel D, Sotta B, Miginiac E (1999) Induction of RAB18 gene expression and activation of K+ outward-rectifying channels depend on an extracellular perception of ABA in Arabidopsis thaliana suspension cells. Plant J 18: 13–22
    OpenUrlCrossRefPubMed
  24. ↵
    Jouanneau JP, Péaud-Lenoël C (1967) Growth and synthesis of proteins in cell suspensions of kinetin dependent tobacco. Physiol Plant 20: 834–850
    OpenUrlCrossRef
  25. ↵
    Katagiri T, Ishyama K, Tabata S, Kobayashi M, Shinozaki K (2005) An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana. Plant J 43: 107–117
    OpenUrlCrossRefPubMed
  26. ↵
    Lang V, Mäntylä E, Welin B, Sundberg B, Palva ET (1994) Alterations in water status, endogenous abscisic acid content, and expression of rab18 gene during the development of freezing tolerance in A. thaliana. Plant Physiol 104: 1341–1349
    OpenUrlAbstract
  27. ↵
    Lang V, Palva ET (1992) The expression of rab-related gene RAB18 is induced by abscisic acid during cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Mol Biol 20: 951–996
    OpenUrlCrossRefPubMed
  28. ↵
    Leonhardt N, Kwak J, Robert N, Waner D, Leonhardt G, Schroeder J (2004) Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16: 596–615
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Lepage M (1967) Identification and composition of turnip root lipids. Lipids 2: 244–250
    OpenUrlCrossRefPubMed
  30. ↵
    Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49: 199–222
    OpenUrlCrossRefPubMed
  31. ↵
    Levchenko V, Konrad K, Dietrich P, Roelfsema M, Hedrich R (2005) Cytosolic abscisic acid activates guard cell anion channels without preceding Ca2+ signals. Proc Natl Acad Sci USA 102: 4203–4208
    OpenUrlAbstract/FREE Full Text
  32. ↵
    MacRobbie E (2000) ABA activates multiple Ca2+ fluxes in stomatal guard cells, triggering vacuolar K+ (Rb+) release. Proc Natl Acad Sci USA 97: 12361–12368
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Marmagne A, Rouet MA, Ferro M, Rolland N, Alcon C, Joyard J, Garin J, Barbier-Brygoo H, Ephritikhine G (2004) Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome. Mol Cell Proteomics 3: 675–691
    OpenUrlAbstract/FREE Full Text
  34. ↵
    McAinsh M, Browlee A, Hetherington A (1990) Abscisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stomatal closure. Nature 343: 186–188
    OpenUrlCrossRef
  35. ↵
    Meijer HJG, Munnik T (2003) Phospholipid-based signaling in plants. Annu Rev Plant Biol 54: 265–306
    OpenUrlCrossRefPubMed
  36. ↵
    Munnik T, Arisz SA, de Vrije T, Musgrave A (1995) G-protein activation stimulates phospholipase D signaling in plants. Plant Cell 7: 2197–2210
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Munnik T, de Vrije T, Irvine RF, Musgrave A (1996) Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants. J Biol Chem 271: 15708–15715
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Munnik T, Meijer H, ter Riet B, Hirt H, Frank W, Bartels D, Musgrave A (2000) Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate. Plant J 22: 147–154
    OpenUrlCrossRefPubMed
  39. ↵
    Nylander M, Svensson J, Palva T, Welin B (2001) Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant Mol Biol 45: 263–279
    OpenUrlCrossRefPubMed
  40. ↵
    Oshiro J, Han GS, Carman G (2003) Diacylglycerol pyrophosphate phosphatase in Saccharomyces cerevisiae. Biochim Biophys Acta 1635: 1–9
    OpenUrlCrossRefPubMed
  41. ↵
    Pical C, Westergren T, Dove S, Larsson C, Sommarin M (1999) Salinity and hyperosmotic stress induce rapid increases in phosphatidyl 4,5-bisphosphate, diacyglycerol pyrophosphate and phosphatidylcholine in Arabidopsis thaliana cells. J Biol Chem 274: 38232–38240
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Pierrugues O, Brutesco C, Oshiro J, Gouy M, Deveaux Y, Carman G, Thuriaux P, Kazmaier M (2001) Lipid phosphate phosphatase in Arabidopsis. J Biol Chem 276: 20300–20308
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Ramanjulu S, Bartels D (2002) Drought- and desiccation-induced modulation of gene expression in plants. Plant Cell Environ 25: 141–151
    OpenUrlCrossRefPubMed
  44. ↵
    Razem F, El-Kereamy A, Abrahms S, Hill R (2006) The RNA-binding protein FCA is an abscisic acid receptor. Nature 439: 290–294
    OpenUrlCrossRefPubMed
  45. ↵
    Riera M, Valon C, Fenzi F, Giraudat J, Leung J (2005) The genetics of adaptative responses to drought stress: abscisic acid-dependent and abscisic acid-independent signalling components. Physiol Plant 123: 111–119
    OpenUrlCrossRef
  46. ↵
    Ritchie S, Gilroy S (1998) Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity. Proc Natl Acad Sci USA 95: 2697–2702
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Roberts JK, DeSimone NA, Lingle WL, Dure L III (1993) Cellular concentrations and uniformity of cell-type accumulation of two LEA proteins in cotton embryos. Plant Cell 5: 769–780
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Schroeder J, Hagiwara S (1989) Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338: 427–430
    OpenUrlCrossRef
  49. ↵
    Schwarz M, Schroeder J (1998) Abscisic acid maintains S-type anion channel activity in ATP-depleted Vicia faba guard cells. FEBS Lett 428: 177–182
    OpenUrlCrossRefPubMed
  50. ↵
    Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14: 194–199
    OpenUrlCrossRefPubMed
  51. ↵
    Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3: 217–223
    OpenUrlCrossRefPubMed
  52. ↵
    Skriver K, Mundy J (1990) Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2: 503–512
    OpenUrlFREE Full Text
  53. ↵
    Swire-Clark G, Marcotte W (1999) The wheat LEA protein Em functions as an osmoprotective molecule in Saccharomyces cerevisiae. Plant Mol Biol 39: 117–128
    OpenUrlCrossRefPubMed
  54. ↵
    Testerink C, Munnik T (2005) Phosphatidic acid: a multifunctional stress signaling lipids in plants. Trends Plant Sci 10: 368–375
    OpenUrlCrossRefPubMed
  55. ↵
    van der Luit A, Piatti T, van Doorn A, Musgrave A, Felix G, Boller T, Munnik T (2000) Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerolpyrophosphate. Plant Physiol 123: 1507–1515
    OpenUrlAbstract/FREE Full Text
  56. ↵
    van Schooten B, Testerink C, Munnik T (2006) Signalling diacylglycerol pyrophosphate, a new phosphatidic acid metabolite. Biochim Biophys Acta 11761: 151–159
    OpenUrl
  57. ↵
    Vergnolle C, Vaultier M-N, Taconnat L, Renou J-P, Kader J-C, Zachowski A, Ruelland E (2005) The cold-induced early activation of phospholipase C and D pathways determine the response of two distinct clusters of genes in Arabidopsis cell suspensions. Plant Physiol 139: 1217–1233
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Verwoerd T, Dekker B, Hoekema A (1989) A small-scale procedure for rapid isolation of plant RNA. Nucleic Acids Res 17: 2362
    OpenUrlFREE Full Text
  59. ↵
    Wang X (2001) Plant phospholipases. Annu Rev Plant Physiol Plant Mol Biol 52: 211–213
    OpenUrlCrossRefPubMed
  60. ↵
    Welin B, Olson Å, Nylander M, Palva ET (1994) Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol Biol 26: 131–144
    OpenUrlCrossRefPubMed
  61. ↵
    Wendehenne D, Lamotte O, Frachisse J-M, Barbier-Brygoo H, Pugin A (2002) Nitrate efflux is an essential component of the cryptogein signaling pathway leading to defense responses and hypersensitive cell death in tobacco. Plant Cell 14: 1937–1951
    OpenUrlAbstract/FREE Full Text
  62. ↵
    Wise M, Tunnacliffe A (2004) POPP the question: What do LEA proteins do? Trends Plant Sci 9: 13–17
    OpenUrlCrossRefPubMed
  63. ↵
    Wissing JB, Behrbohm H (1993a) Diacyglycerol pyrophosphate, a novel phospholipid compound. FEBS Lett 315: 95–99
    OpenUrlCrossRefPubMed
  64. ↵
    Wissing JB, Behrbohm H (1993b) Phosphatidate kinase, a novel enzyme in phospholipid metabolism: purification, subcellular localization, and occurrence in the plant kingdom. Plant Physiol 102: 1243–1249
    OpenUrlAbstract
  65. ↵
    Wissing JB, Kornak B, Funke A, Riedel B (1994) Phosphatidate kinase, a novel enzyme in phospholipid metabolism. Plant Physiol 105: 903–909
    OpenUrlAbstract
  66. ↵
    Wu WI, Liu Y, Riedel B, Wissing JB, Fischl AS, Carman GM (1996) Purification and characterization of diacyglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae. J Biol Chem 271: 1868–1876
    OpenUrlAbstract/FREE Full Text
  67. ↵
    Zalejski C, Zhang Z, Quettier A-L, Maldiney R, Bonnet M, Brault M, Demandre C, Miginiac E, Rona J-P, Sotta B, et al (2005) Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cells. Plant J 42: 145–152
    OpenUrlCrossRefPubMed
  68. ↵
    Zhang W, Qin C, Zhao J, Wang X (2004) Phospholipase Dα1-derivated phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling. Proc Natl Acad Sci USA 101: 9508–9513
    OpenUrlAbstract/FREE Full Text
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Induction of Abscisic Acid-Regulated Gene Expression by Diacylglycerol Pyrophosphate Involves Ca2+ and Anion Currents in Arabidopsis Suspension Cells
Christine Zalejski, Sophie Paradis, Régis Maldiney, Yvette Habricot, Emile Miginiac, Jean-Pierre Rona, Emmanuelle Jeannette
Plant Physiology Aug 2006, 141 (4) 1555-1562; DOI: 10.1104/pp.106.080218
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