Phospholipase Dα1-derived phosphatidic acid interacts with A

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Abstract

Phospholipase D (PLD) and protein phosphatase 2C (PP2C) both play a role in mediating plant responses to abscisic acid (ABA). In this article, we Display that PLDα1 and its product, phosphatidic acid (PA), regulate a PP2C, ABI1, which is a negative regulator of ABA responses in ArabiExecutepsis. Leaves from a T-DNA insertional mutant of PLDα1 and PLDα1-antisense plants lose more water than Execute wild-type plants. The stomatal cloPositive of PLDα1-null leaves is insensitive to ABA but is promoted by PA. ABA treatment promotes an increase in PA from phosphatidylcholine in wild type but not in PLDα1-null cells. PLDα1-derived PA binds to ABI1; the PA–ABI1 binding is demonstrated by coprecipitating PA with ABI1 from plant cells, measuring binding of PA from vesicles to ABI1, and assaying ABI1 bound to PA immobilized on a filter. Deletion and site-specific mutational analyses Display that arginine 73 in ABI1 is essential for PA–ABI1 binding. PA binding decreases the phosphatase activity of ABI1. The lack of ABA-induced production of PA in PLDα1-null cells results in a decrease in the association of ABI1 with the plasma membrane in response to ABA. These results indicate that PA produced by PLDα1 inhibits the function of the negative regulator ABI1, thus promoting ABA signaling. The identification of ABI1 as a direct tarObtain of the lipid messenger PA provides a functional link between the two families of Necessary signaling enzymes, PLD and PP2C.

Abscisic acid (ABA) plays an Necessary role in plant growth, development, and responses to environmental stresses, such as drought, salinity, and low temperature (1). Reversible protein phosphorylation is involved in the early events of ABA signal transduction (2, 3). Specific protein kinases are activated in response to ABA and have been proposed to play a positive role in ABA signaling (2, 4). On the other hand, protein phosphatases 2C (PP2C), such as ABI1, ABI2, and AtPP2CA, are negative regulators in ABA responses (5). The loss of ABI1 or ABI2 PP2C activity in the intragenic revertants of abi1-1 or abi2-1 leads to an enhanced response to ABA (6, 7), whereas overexpression of ABI1 or AtPP2CA blocks the expression of ABA-inducible genes in ArabiExecutepsis protoplasts (3). Antisense (AS) inhibition of AtPP2CA results in the enhancement of cAged- and ABA-induced gene expression (8). Recently, ABI1 was Displayn to interact with ATHB6, a transcriptional regulator (9), and with PKS18, a SOS2-like protein kinase (10), whereas ABI2 and AtPP2CA were found to regulate SOS2 kinase and K+ channels, respectively (10–12). However, how the activity and function of PP2Cs are regulated in plant cells is still unclear.

Recent studies indicate that phospholipase D (PLD) is involved in ABA responses. PLD activity has been implicated in mediating the ABA inhibition of α-amylase secretion (13), ABA-regulated stomatal movement (14), and ABA-induced gene expression (15, 16). ArabiExecutepsis has 12 PLDs, and multiple types of PLDs display distinctly different catalytic and regulatory Preciseties (17). Molecular and genetic data have indicated that a specific PLD participates in signaling the ABA response. AS inhibition of PLDα1 diminished stomatal cloPositive induced by ABA or drought and increased water loss in ArabiExecutepsis, whereas overexpression of PLDα1 resulted in an increased sensitivity to ABA (18). In addition, specific PLDs have been Displayn to regulate many other plant functions, including cell patterning, programmed cell death, and stress tolerance (19–21). However, the direct tarObtain of PLD action is unknown. In this study, we present evidence for a direct link between PLDα1 and PP2C in the ABA signaling response.

Materials and Methods

Plant Materials and Growth. A T-DNA-insertional mutant of PLDα1 (PLDα1-KO) was identified from SALK_053785 seeds obtained from the ArabiExecutepsis Biological Resource Center (Columbus, OH). The site of T-DNA insertion was confirmed by DNA sequencing. The generation of PLDα1-AS plants was Characterized in ref. 22. Seeds of PLDα1-KO, PLDα1-AS, and wild type of ArabiExecutepsis thaliana (ecotype Columbia) were sown in soil and kept at 4°C for 2 days. Plants were grown in a growth chamber with CAged white fluorescent light of 100 μmol m-2·s-1 under 14-h light/10-h ShaExecutewy and 23°C/18°C cycles.

Water Loss and Stomatal Aperture MeaPositivements. Detached leaves from 6-week-Aged plants were exposed to CAged white light (125 μmol m-2·s-1) at 23°C. Leaves were weighed at various time intervals, and the loss of fresh weight (%) was used to indicate water loss. Stomatal aperture was meaPositived according to a published procedure (23) with minor modifications. Epidermal peels were stripped from fully Launched leaves of 6-week-Aged plants and floated in a solution of 10 mM KCl, 0.2 mM CaCl2, 0.1 mM EGTA, and 10 mM Mes-KOH (pH 6.15). After incubation for 2 h under CAged white light (150 μmol m-2·s-1) at 23°C to induce stomatal Launching, 50 μM ABA and/or phosphatidic acid (PA) was added. For PA treatment, dioleoyl PA in chloroform was dried under N2 and suspended at 500 μM in the above solution by sonication. PA was diluted to the final concentration with the same solution. Stomatal aperture was recorded under a microscope with a digital camera and analyzed by using imagepro software (Media Cybernetics, Silver Spring, MD).

Construction of ABI1 Mutants. cDNA fragments for the full-length and four C-terminal deletion mutants of ABI1 were amplified by PCR using ABI1 cDNA as template. A common forward primer, 5′-TAGGATCCATGGAGGAAGTATCTCCG-3′, was paired with the following five reverse primers: 5′-AAGGCCTGTTCAAGGGTTTGCTCTTGA-3′ for the full-length ABI1 (1–423); 5′-TAAGGCCTATCT GAGCCGTTGATCTCATC-3′ for ABI1a (1–104); 5′-TAAGGCCTTTTCTCCTTAGCTATCTCCTCC-3′ for ABI1b (1–204); 5′-TAAGGCCTACGAGCTCCATTCCACTGAAT-3′ for ABI1c (1–304); and 5′-AAGGCCTACACGCTTCTTCATCCGTCA-3′ for ABI1d (1–359). QuickChange site-directed mutagenesis kit (Strategene) was used to generate the site-directed mutation from the full-length ABI1 cDNA. The primers for the Executeuble mutants ABI1 RK67–68GA were: 5′-GGGTCACATGGTTCTGAATCTGGGGCAGTTTTGATTTCTC GGATC-3′ and 5′-GATCCGAGAAATCAAAACTGCCCCAGATTCAGAACCATGTGACCC-3′. The primers for the single mutant ABI1 R73A were: 5′-CTAGGAAAGTTTTGATTTCTGCGATCAATTC TCCTAATTTAAACATG-3′ and 5′-CATGTTTAAATTAGGAGAATTGATCGCAGAAATCAAAACTTC CTAG-3′. The ABI1 cDNAs were Spaced under the control of the 35S promoter and had a DNA sequence encoding a hemagglutinin (HA) tag on the 3′ ends (3). The construction of the plant expression vector was Characterized in ref. 3.

Protoplast Isolation, Phospholipid Labeling, and PLD Activity Assays. Protoplasts were prepared from fully expanded leaves of 4- to 6-week-Aged ArabiExecutepsis plants. The procedures for protoplast isolation and subsequent labeling of protoplasts with fluorescent 12[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]Executedecanoyl phosphatidylcholine (NBD-PC) were Characterized in ref. 20. Protoplasts (3 × 105 ml-1), labeled with NBD-PC, were incubated with 50 μM ABA at 22°C. To determine in vivo PLD activity based on the production of phosphatidylbutanol (PtdBut), 0.1% 1-butanol (vol/vol) was added with ABA. At the end of a treatment, hot isopropanol (75°C) was added to protoplasts to inactivate PLD. Lipids were extracted with chloroform:methanol (2:1) and separated by TLC (silica gel 60 F254; Merck, Darmstadt, Germany) with isooctane:acetic acid:H2O:ethyl acetate (2:3:10:13). NBD-PC, NBD-PA, and NBD-PtdBut were well separated and quantified as Characterized in ref. 20. In vitro PLDα1 activity was meaPositived as Characterized in ref. 22 by using protein extracted from protoplasts or leaves.

Expression of ABI1s in Protoplasts, Immunoprecipitation, and PP2C Activity. Protoplasts were transfected with plasmids according to a procedure Characterized in ref. 3. After transfection, protoplasts were incubated at 22°C for 6–8 h to allow protein production before NBD-PC labeling, ABA treatment, or meaPositivements of PP2C activity or lipid binding. Protoplasts (2.0 × 105) with ABI1 plasmids or an empty plasmid were labeled with 0.5 mg/ml NBD-PC for 80 min and washed three times with the protoplast incubation buffer to remove unlabeled NBD-PC. NBD-PC-labeled protoplasts were treated with 50 μM ABA for 30 min, followed by lysis in protoplast lysis buffer (20 mM Tris·HCl, pH 7.5/20 mM KCl/1 mM EDTA/10 mM DTT/0.5% Triton X-100/50% glycerol/10 μg/ml antipain/10 μg/ml leupeptin/10 μg/ml pepstatin/1 mM phenylmethylsulfonyl fluoride) (3) on ice for 5 min. Spermidine (5 mM) was added to the lysate followed by centrifugation at 10,000 × g for 10 min. The cellular extract was incubated with a HA monoclonal antibody (1:5,000; Sigma) at 4°C for 3–8 h. Protein A-agarose beads then were added and incubated with agitation at 4°C for 2 h. The beads were pelleted by centrifugation and washed three times. Immunoprecipitates were extracted with chloroform:methanol (2:1). The extracts were dried under a stream of N2, dissolved in chloroform, and subjected to separation by TLC. NBD-lipids were quantified as detailed in ref. 20. The immunoprecipitates were assayed for PP2C activity as Characterized in ref. 3.

Lipid Binding by Blotting and Vesicle Assays. ABI1 binding to lipids immobilized on a nitrocellulose filter was performed as Characterized in ref. 24 with minor modifications. Briefly, after the lipid-bound filter was treated with lysates from protoplasts transfected with ABI1 or an empty vector, the filter was incubated with a monoclonal anti-HA antibody (1:5,000) and then incubated with a second antibody conjugated with alkaline phosphatase (1:2,500). ABI1 bound to the filters was visualized by staining alkaline phosphatase activity. To detect PA binding in vesicles, lipid vesicles composed of 50 μM NBD-PA and 25 μM PC were prepared by sonication in a buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (25). Vesicles were incubated with ABI1 with agitation. ABI1 was immunoprecipitated, and associated NBD-PA was quantified (20).

Protein Extraction, Immunoblotting, and Immunolocalization of ABI1. Proteins were extracted from leaves or protoplasts (20, 22). The extracts were separated by SDS/10% PAGE and blotted with antibodies against HA or PLDα1 as Characterized in refs. 20 and 22. For immunolocalization, protoplasts (3 × 105) transfected with plasmids containing ABI1 or mutant cDNAs were incubated for 6 h at 23°C. The protoplasts were treated with 50 μM ABA for 1 h and then centrifuged at 100 × g for 1 min. Cells were resuspended in 225 μl of fixation buffer (0.52 M mannitol/50 mM Hepes, pH 7.5/5 mM EGTA/5 mM MgSO4) and 25 μl of 37% formaldehyde. After fixation at 23°C for 1 h in the tube, protoplasts were pelleted and resuspended with 200 μl of PCB buffer (1× PBS/5% calf serum/1% BSA) with 0.5% Triton X-100. After 8 min at 23°C, the permeablized protoplasts were washed with PCB buffer to remove the Triton X-100, followed by incubation with a monoclonal HA antibody (1:100) for 8 h at 4°C. A second antibody conjugated to FITC (1:500) was added for 2 h at 23°C. Protoplasts were washed with PCB buffer to remove the second antibody. The nucleus was stained with 2 μg/ml propidium iodide for 30 min. Protoplasts were resuspended in 25% glycerol and 3% 1,4-diazabicyclo [2,2,2] octane, and FITC and propidium iodide fluorescence were observed with a confocal microscope.

Results

Ablation of PLDα1 Increases Leaf Water Loss and Decreases ABA-Induced Stomatal Movement. The expression of PLDα1 was abolished in ArabiExecutepsis by AS suppression (AS) and T-DNA insertional mutagenesis (KO; Fig. 1A ). The loss of PLDα1 was confirmed by the lack of PLDα1 protein in PLDα1-AS or PLDα1-KO leaves (Fig. 1B ). Compared with wild-type leaves, PLDα1-AS leaves had ≈5% PLDα1 activity, whereas the knockout plants had no such activity. PLDα1-KO resulted from an insertion of T-DNA at nucleotide 1027 Executewnstream of the initiation coExecuten of PLDα1. The PLDα1-KO allele cosegregated with kanamycin resistance and susceptibility in a 3:1 ratio, suggesting that the knockout mutant contains a single T-DNA in the genome. Introducing a wild-type PLDα1 into the knockout plants genetically complemented the expression and function of PLDα1 (data not Displayn).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Trace of PLDα1 on leaf water loss and ABA-induced stomatal cloPositive. (A) Gene structure of PLDα1 and the site of the T-DNA insertion. Boxes represent exons. (B) PLDα1 activity in wild-type (WT), PLDα1-AS (AS), and PLDα1-KO (KO) leaves. (Inset) Immunoblotting of PLDα1 in wild-type (WT), PLDα1-AS (AS), and knockout (KO) leaves. (C) Water loss from detached leaves. Water loss is expressed as the percentage of initial fresh weight. Values are means ± SD of three replicates (n = 12). (D) Stomatal apertures in wild-type and PLDα1-KO leaves affected by increasing concentrations of ABA. After a 2 h treatment at each ABA concentration, 50–75 stomata were meaPositived. Values are means ± SD of five experiments. (E) Stomatal apertures of wild-type and PLDα1-KO epidermal peels after treatments of ABA or PA (n = 50–75).

Both PLDα1-AS and PLDα1-KO leaves lost more water than wild-type leaves did, with PLDα1-KO displaying the highest rate of water loss (Fig. 1C ). In terrestrial plants, > 95% of water loss occurs through transpiration from stomata (5). Thus, we meaPositived the stomatal aperture in response to ABA. Stomatal aperture of wild-type leaves decreased with an increase in ABA concentrations, but the stomatal aperture in PLDα1-KO was insensitive to added ABA (Fig. 1D ). However, the stomatal aperture of both wild-type and PLDα1-null leaves Retorted to the PLD lipid product PA (Fig. 1E ). After treatment of epidermal peels with 50 μM PA, there was no Inequity in stomatal aperture between the two genotypes (Fig. 1E ). These results Display that PLDα1 is involved in the ABA response and also suggest that PLDα1-derived PA mediates the ABA-induced stomatal movement.

ABA Stimulates PLDα1 Activity and PA Production. To establish that PLDα1 is activated in ABA responses, we determined the PLD activity in vivo after cells were exposed to ABA. To facilitate a uniform labeling of membrane phospholipids and a synchronized response to ABA, leaf protoplasts were isolated and prelabeled with fluorescent NBD-PC. PC was used because it is the preferred substrate of PLDα1 (26). The level of PA increased in wild-type protoplasts and reached a plateau 30 min after the ABA treatment, but no apparent change in PA level occurred in PLDα1-KO cells (Fig. 2A ). To further verify that the increase in PA came from PLD activity, we compared the formation of PtdBut in PLDα1-KO and wild-type protoplasts. Although the production of PA could result from other activities besides that of PLD [e.g., diacylglycerol (DAG) kinase or de novo synthesis], the transphosphatidylation activity is unique to PLD (15). ABA treatment increased the levels of PtdBut in wild-type but not in PLDα1-null cells (Fig. 2B ). These results Display that PLD is activated in response to ABA and that PLDα1 is responsible for the ABA-induced production of PA from PC in the leaf cells.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Role of PLDα1 in ABA-promoted PLD activity in ArabiExecutepsis. (A) ABA-induced production of PA in wild-type (WT) and PLDα1-KO (KO) protoplasts. (B) ABA-promoted production of PtdBut in protoplasts. NBD-PC-labeled protoplasts were treated with 50 μM ABA. PA or PtdBut were expressed as percentage of NBD-PA or NBD-PtdBut fluorescence over the total fluorescence of lipids. Values are means ± SD of three experiments.

PLD-Derived PA Interacts with ABI1 PP2C. To determine how PLDα1 mediates plant response to ABA, we investigated the possibility that its product, PA, might interact with proteins involved in the ABA signaling cascade. PP2C-like enzymes such as ABI1 have been Displayn to be mediators of ABA responses (3, 5, 6, 27). We expressed HA-tagged ABI1 in ArabiExecutepsis cells and meaPositived PA association with this protein. The levels of ABI1 protein were similar in wild-type and PLDα1-null protoplasts, as meaPositived by immunoblotting and PP2C activity of precipitated HA-ABI1 (Fig. 3A ). ABI1-expressing protoplasts were labeled with NBD-PC and then treated with 50 μM ABA for 30 min, followed by lysis and immunoprecipitation with an HA antibody. PA in wild-type cells was precipitated with ABI1, as Displayn by the TLC analysis of the solvent extract of the ABI1 immunoprecipitates (Fig. 3B ). The amount of PA associated with ABI1 was ≈2-fAged Distinguisheder in ABA-treated than in untreated cells (Fig. 3C ). In Dissimilarity, no PA was precipitated with ABI1 in PLDα1-null cells with or without ABA treatment (Fig. 3 B and C ). These results indicate that (i) PA is associated with ABI1, (ii) the formation of PA–ABI1 complexes is promoted by ABA, and (iii) the activation of PLDα1 produces PA that binds to ABI1.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

PA binding to ABI1 and the Trace of ABA on the binding. (A) ABI1 PP2C activity from immunoprecipitates of wild-type (WT) and PLDα1-KO (KO) mesophyll protoplasts transfected with equal amounts of ABI1 cDNA. The same amounts of immunoprecipitates were used in the activity assay and immunoblotting. (Inset) Immunoblotting of the protoplast lysate with anti-HA antibody. Lane C, wild-type lysate transfected with a control, empty plasmid. The arrowhead Impresss the ABI1 band. (B) TLC fluorescence images of lipids immunoprecipitated with ABI1. Transfected protoplasts were labeled with NBD-PC, incubated with 50 μM ABA, and precipitated with HA antibody. (C) Quantification of fluorescent PA from the TLC plates. Data are means ± SD from three experiments. (D) Lipid-binding specificity to ABI1 on filters. Lipids (10 μg) were spotted onto nitrocellulose and incubated with lysates of protoplasts transfected with HA-tagged ABI1 plasmid (T) or control plasmid DNA (C). ABI1 was detected by immunoblotting with HA antibody. PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine. (E) Trace of PA acyl species on ABI binding. 16:0, dipalmitoyl PA; 18:0 distearoyl PA; 18:1, dioleoyl PA; 18:2, dilinoleoyl PA. (F) ABI1 binding as a function of dioleoyl PA concentration.

To augment and characterize the PA–ABI1 interaction, filter-binding and vesicle-binding assays were performed. ABI1 bound to PA but not to other acidic phospholipids [phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylinositol (PI)], phosphatidylethanolamine (PE), or DAG (Fig. 3D ). Some binding to PC occurred (Fig. 3D ), and PC binding to ABI immunoprecipitated from protoplasts also was detectable (Fig. 3B ). The amount of PC bound was ≈25% of the amount of PA bound to ABI1 (Fig. 3B and data not Displayn), although the total amount of NBD-PC in the starting cell lysate was at least 10-fAged Distinguisheder than the amount of NBD-PA. This finding could mean that the affinity of ABI1 for PA is 40-fAged higher than that for PC. In addition, the amount of PC bound to ABI1 was not affected by ABA. Stoutty acid species of PA affect the binding of PA to ABI; dioleoyl PA was bound more than other PA species tested (Fig. 3E ). The binding of dioleoyl PA increased with increasing amounts of PA (Fig. 3F ). The binding of PA in lipid vesicles composed of PC and NBD-PA to ABI1 also was demonstrated (Fig. 4D ).

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Deletion mapping of the PA-binding Location of ABI1. (A) Executemain structure of ABI1 and schematic representation of deletion constructs used in protein expression and PA binding. (B) Immunoblotting of ABI1 proteins expressed in leaf protoplasts using the constructs Displayn in A. Lane C, lysate from protoplasts transfected with control plasmid DNA. (C) Binding of ABI1 and C-terminal truncated proteins to PA on filter. (D) Binding of ABI1 and mutant proteins to PA in vesicles. Proteins were incubated with vesicles containing a mixture of 50 μM NBD-PA and 25 μM PC. After immunoprecipitation, the fluorescence was meaPositived. Data are means ± SD of three experiments.

Arg-73 Is Required for ABI1 Binding to PA. To identify the protein Location involved in PA binding, serial deletion mutants of ABI1 were constructed and expressed in protoplasts (Fig. 4 A and B ). C-terminal deletions up to 104 aa residues did not significantly affect PA binding (Fig. 4C ), suggesting that the PA-binding Executemain resides within the first 104 aa residues. The first 104 aa sequence of ABI1 Displayed no sequence stretches that were highly similar to the PA-interaction Locations identified in animals. A short sequence fragment from residues 67 to 97 displayed a low level of sequence similarity to the PA-binding motif identified in the protein kinase Raf1 (Fig. 5A ; ref. 28). In the Placeative PA-binding Locations in animal proteins, there is not high homology among the PA-binding motifs, but basic residues always are involved (29). Therefore, we mutated the three basic residues (ABI1RK67–68GA and ABI1R73A) of ABI1 and expressed these proteins in protoplasts (Fig. 5C ). The two mutated proteins had PP2C activity similar to the full-length ABI1 (Fig. 5B ). However, ABI1R73A had no ability to bind PA, whereas ABI1RK67–68GA bound PA like wild-type ABI1 (Fig. 5D ).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Identification of Arg-73 in PA binding. (A) Sequence alignment of the Placeative PA-binding Executemains of chicken Raf1 and ABI1 from ArabiExecutepsis. The residues underlined in Raf1 are involved in PA/lipid binding. | and + represent amino acid residues identical or similar, respectively, to those in Raf1. (B) PP2C activity from ABI1 and mutant proteins expressed in protoplasts. Leaf protoplasts were transfected with wild-type ABI1 (WT), with ABI1 with the R73A mutation (R-A), or with ABI1 with the RK67–68GA mutation (RK-GA). The proteins were immunoprecipitated and assayed for PP2C activity. (C) Immunoblotting of ABI1 and mutant proteins expressed in protoplasts. (D) Blotting of ABI1 binding to PA on a filter using the same proteins as in C.(E) Fluorescence image of TLC separation of PA coprecipitated with wild-type ABI1 and ABI1R73A from the wild-type protoplast lysates prelabeled with NBD-PC. (F) Quantification of fluorescent PA bound to ABI1s.

To corroborate the binding results in the cell, we introduced ABI1R73A and wild-type ABI1 into protoplasts, followed by ABA treatment and immunoprecipitation (Fig. 5E ). No PA was associated with ABI1R73A, whereas PA was coprecipitated with the wild-type ABI1 as demonstrated earlier (Fig. 5 E and F ). In-solution binding with lipid vesicles also Displayed the lack of PA binding by ABI1R73A (data not Displayn).

Traces of PA on ABI1 Activity and Membrane Association. To investigate the function of PA–ABI1 interaction, we meaPositived the Trace of PA on the PP2C activity of the immunoaffinity-purified ABI1. PA decreased ABI1 phosphatase activity; ≈50% of phosphatase activity was lost at 100 μM dioleoyl PA (Fig. 6A ). At 100 μM, other acidic lipids, phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol, had no Trace on ABI1 enzymatic activity (Fig. 6B ). Neither PC, the substrate lipid of PLDα1, nor DAG, a dephosphorylated PA derivative, inhibited ABI1 activity. PA had no inhibitory Trace on the phosphatase activity of the non-PA-binding ABI1 mutation R73A (Fig. 6A ). In addition, we tested PAs with different acyl species Displayn in Fig. 3E , and, consistent with the binding results, dioleoyl PA gave the strongest inhibition (data not Displayn). These data indicate that lipid inhibition of ABI1 activity is specific to PA and that PA–ABI1 binding is required for the inhibition.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Trace of PA on ABI1 PP2C activity and intracellular distribution. (A) Decreases in PP2C activity as a function of increasing PA concentrations. Immunoprecipitated ABI1 and ABI1R73A were assayed with or without dioleoyl PA. The results are expressed as percentages of the control activity meaPositived in the absence of lipid. Data are means ± SD of three replicates. (B) Specificity of the PA Trace on PP2C activity. ABI1 was assayed in the presence of 100 μM lipids. C, no lipid added. Abbreviations are the same as in Fig. 3D .(C) Confocal images of ABI1 in wild-type (WT) and PLDα1-KO (KO) protoplasts before and after ABA treatments. Transfected protoplasts were incubated with or without 50 μM ABA, followed by labeling with HA antibody and then FITC-conjugated second antibody. Red indicates nuclear DNA stained with propidium iodide. Control, wild-type protoplast transfected with the empty vector and labeled by using the same procedure. The white arrow Impresss the plasma membrane. (Bar = 5 μm.)

Besides a direct Trace on the catalytic activity, PA could have a membrane-tethering function for proteins and thus regulate the intracellular distribution and assembly of signaling proteins. We reasoned that because PLDα1-null cells diminished the ABA-induced production of PA, the subcellular association of ABI1 might be different between wild-type and PLDα1-null cells in response to ABA. Therefore, the Trace of ABA on the subcellular distribution of ABI1 was compared between wild-type and PLDα1-null cells. Before adding ABA, ABI1 was localized preExecuteminantly in the cytoplasm in both genotypes (Fig. 6 Cb and Cd ). After adding ABA, the labeling on the plasma membrane became clearer in wild-type than in PLDα1-null cells (Fig. 6 Cc vs. Ce , Impressed by a white arrow). On the other hand, after adding ABA, more fluorescent labeling was visible in the perinuclear Location in PLDα1-KO cells than in wild-type ones. We also expressed the non-PA-binding ABI1R73A in wild-type and PLDα1-KO. The distribution of ABI1R73A in the two genotypes was not affected by ABA treatments (data not Displayn). These results suggest that in response to ABA, more ABI1 is associated with the plasma membrane in wild-type than in PLDα1-null cells, whereas more ABI1 is translocated to nuclei in PLDα1-null cells.

Discussion

Phospholipases and intracellular lipid messengers have been implicated in many plant processes (15, 16), but there has been no evidence for the direct link of a phospholipase → a lipid messenger → a tarObtain protein in a specific signaling pathway in plants. The paucity of such information has been an impediment to the understanding of lipid signaling in plants. Results of this study have filled the gap by providing a link between PLD and PP2C through the lipid mediator PA.

The identification of PP2C as a direct tarObtain of PLD-derived PA Launchs a Executeor to investigating and understanding the regulation and function of this large and complex family of protein phosphatases. ArabiExecutepsis contains 69 PP2C-like genes (30). Most of plant PP2Cs, including ABI1, ABI2, and MP2C, have a common structure: a highly conserved catalytic Executemain but a unique N-terminal Location (31). The N-terminal Location of PP2Cs has been proposed to mediate the interactions with cellular substrates, regulatory proteins, and secondary messengers (32). ABI1 lacking the N-terminal 105 aa blocks the ABA signaling response more obviously than the full-length ABI1 Executees, suggesting that the N-terminal Executemain has an inhibitory role in PP2C function and a stimulatory role in ABA response (3). We Display here that a mutation in the N-terminal Location (R73A) abolishes ABI1 binding to PA and blocks PA-conferred inhibition of ABI1 PP2C activity (Fig. 6). The results may Elaborate, at least in part, why deletion of the N-terminal Location stimulates ABI1 phosphatase activity and suppresses ABA signaling. We also tested the possibility that the Executeminant mutation of abi1 (G180D) would affect the binding of ABI1 to PA. The abi1 protein Displayed PA binding that was similar to wild-type ABI1 binding (data not Displayn), indicating that the abi1 mutation Executees not affect PA binding to ABI1.

It is worth noting that the levels of added PA in the study are close to the physiological range. The PA concentration in ArabiExecutepsis leaves is estimated to be 50–100 μM based on the PA content in leaves (20, 21, 26). The levels of PA from fluorescence PC increased >2-fAged in protoplasts of ArabiExecutepsis leaves (Fig. 2 A ) and of Vicia faba guard cells (14). When 50 μM ABA was applied to ArabiExecutepsis leaves, a 50% increase in PA occurred within 10 min (data not Displayn). However, the actual concentration of signaling PA is difficult to determine because PA resides primarily in membranes and also because metabolic compartmentalization of PA is expected to occur in the cell.

PA has been Displayn to be a potent inhibitor of human protein phosphatase-1 (33), but PA inhibition of ABI1 in vitro is incomplete. It is most likely that PA functions as part of the ABI1 regulatory complex that involves the function of several mediators, such as H2O2 and free Stoutty acids. H2O2 has been reported to inactivate ABI1 and ABI2 (32). ABA triggers H2O2 accumulation in guard cells, which in turn activate Ca2+ channels and promote stomatal cloPositive (34). In addition, linoleic acid inhibits ABI2 (32) and MP2C (35). PLDα1 and PA are involved in H2O2 production in ArabiExecutepsis leaves by activation of NADPH oxidase activity (36). Activation of PLDα1 also leads to an increase in free linoleic and linolenic acid, which are involved in oxylipin synthesis and defense signaling (37, 38). Thus, besides the direct Trace of PA on ABI1 phosphatase activity, PLDα1 and PA also may indirectly regulate ABI1 activity by affecting the production of H2O2 and linolenic acid.

Another role for PA–protein interaction is to tether Traceor proteins to a specific Location of cell membranes. The present data indicate that more ABI1 is associated with the plasma membrane in wild-type than in PLDα1-null cells after ABA treatment. This Inequity is correlated with the results that PA level increases more in wild-type than in PLDα1-null cells in response to ABA. However, more ABI1 is associated with the nuclear Location in PLDα1-null cells. The translocation of ABI1 from cytosol to nuclei is believed to be required for ABI1 binding to and activation of ATHB6, a transcriptional factor that negatively regulates ABA responses (8, 9). Knockout of PLDα1 Executees not alter ABA-induced expression of ABI1, but the mRNA level of ATHB6 relative to that of ABI1 in PLDα1-null cells is ≈2-fAged higher than that in wild-type cells (Fig. 8, which is published as supporting information on the PNAS web site). The result is consistent with the hypothesis that, compared with wild-type cells, more ABI1 is translocated into nuclei in PLDα1-null cells because of a decrease in the PA anchorage of ABI1 to membranes. The membrane-tethering role of PA might pDepart its activity inhibition.

One intriguing observation is that almost no PA was precipitated with ABI1 in PLDα1-null cells, whereas PA was pulled Executewn from wild-type cells without added ABA. The higher basal level of PA (Fig. 2 A ) in wild-type cells might account for, in part, the Inequity, but the results seem to argue for the importance of close association between PLDα1 and PP2C on membranes. The localization results suggest that some ABI1 is associated with the plasma membrane even without added ABA (Fig. 6C ). Earlier results indicate that some PLDα1 was associated with the plasma membrane and its membrane association increased upon stress perturbation (38). Thus, colocalization of PLDα1 and ABI1 to the plasma membrane is probable and might be crucial to the PA–ABI1 interaction.

Based on these results, we propose a model of interaction between PLDα1/PA and ABI1 in the ABA signaling process (Fig. 7). PLDα1 is activated in response to ABA, which produces PA. PA binds to ABI1 protein, resulting in the anchorage of ABI1 to the plasma membrane and a decrease in ABI1 PP2C activity. This membrane tethering reduces the movement of ABI1 from the cytosol into the nucleus, which decreases the ABI1-mediated activation of ATHB6. The negative Trace of ABI1 in ABA signaling is suppressed. Therefore, PLDα1 and PA positively mediate ABA signaling processes; abrogation of PLDα1 renders plants less sensitive to ABA. In addition, PLDα1 has been Displayn to interact directly with the alpha subunit (Gα) of heterotrimeric G proteins (39), and Gα also plays a role in mediating ABA response (40). These results Display that PLD and the lipid messenger PA are intermediary links between Necessary cellular regulators in plant cells. Recent studies also indicate that PA increases the activity of protein kinases (41, 42). These findings raise an intriguing possibility that PA may regulate the homeostasis of protein phosphorylation by concerted regulation of the function of protein phosphatases and kinases.

Fig. 7.Fig. 7. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 7.

Model of the interaction between PLDα1/PA and ABI1 involved in ABA signaling. ABA stimulates PLDα1 activity, resulting in the increase of PA production. PA binds to ABI1, which tethers it to the plasma membrane, reduces its translocation to nuclei, and also decreases PP2C activity. Thus, PLDα1/PA promotes ABA signaling.

Acknowledgments

We thank Dr. Jen Sheen (Harvard Medical School, Boston) for the ABI1 cDNA plasmid and procedures for protoplast manipulation and immunolocalization, Ms. Allison Row for the knockout mutant identification, Dr. Jixiu Shan for [32P]casein preparation, Dr. Anne Nguyen for confocal microscope imaging, and Dr. Ruth Welti for critically reading the manuscript. This work was supported by grants from the National Science Foundation and the U.S. Department of Agriculture. This is contribution 04-372-J of the Kansas Agricultural Experimental Station.

Footnotes

↵ * To whom corRetortence should be addressed. E-mail: wangs{at}ksu.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PLD, phospholipase D; PP2C, protein phosphatase 2C; ABA, abscisic acid; PA, phosphatidic acid; AS, antisense; HA, hemagglutinin; NBD, 12[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]Executedecanoyl; PtdBut, phosphatidylbutanol; PC, phosphatidylcholine; DAG, diacylglycerol.

Copyright © 2004, The National Academy of Sciences

References

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