EAR1 Negatively Regulates ABA Signaling by Enhancing 2C Protein Phosphatase Activity

Plant Materials and Growth Conditions

In this study, Arabidopsis thaliana plants harboring a gl1 mutation in the Col-0 background were used as the wild type. The seeds were sown on MS medium containing 2% (w/v) sucrose and 0.8% or 1% (w/v) agar, incubated at 4°C for 3 d, and transferred to a light incubator (Philips F17T8/TL841 bulb) under a 22-h-light/2-h-dark cycle with a light intensity of 60 μE m⁻² s⁻¹ at 22°C. For the root-bending assays, 4-d-old seedlings were transferred to MS medium containing different concentrations of ABA and grown for an additional 7 d. For the germination assays, ∼30 seeds were sown on MS medium containing different concentrations of ABA. The plates were transferred from a temperature of 4°C to the light incubator, where they were grown for 7 d to examine the seed germination ratio, with three plates prepared per experiment. For physiological experiments, the seedlings were grown in pots containing a mixture of forest soil:vermiculite (1:1) under a 12-h-light/12-h-dark cycle in a greenhouse for 3 or 4 weeks.

A T-DNA line, SALK_057606 (ear1-2), with T-DNA inserted at 1088 bp of the AT5G22090 gene, was obtained from the Arabidopsis Biological Resource Center. A homozygous T-DNA insertion line was obtained using PCR. The ABA phenotypes of ear1-2 (the progeny of a cross between ear1-1 and ear1-2) were analyzed.

The other mutants used in this study included abi1-2 (SALK_072009), abi2-2 (SALK_015166), hab1-1 (SALK_002104), ost1/snrk2.6 (SALK_008068), snrk2.2 (GABI-Kat 807G04), and snrk2.3 (SALK_107315). The pp2c triple mutant (3m) was established by crossing abi1-2, abi2-2, and hab1-1, and the 4m mutant was established by crossing 3m and ear1-1. The primers used to examine these mutants are listed in Supplemental Data Set 4.

Map-Based Cloning and Mutant Complementation

The ear1-1 mutant was crossed with Landsberg erecta to obtain the F2 population for selection of mutant plants based on increased ABA sensitivity in roots on MS medium containing 30 μM ABA. Approximately 2819 ear1-1 seedlings were chosen to map EAR1 using SSLP markers. EAR1 was initially mapped to chromosome 5 between BAC clones F8L15 and MOP9, and the locus was then narrowed down to T10F18 and MWD9 using the markers MPI7, F5O24, F22D1, T10F18, T6G21, and MWD9. All genes in this region were sequenced, and only one mutation, C163 to T163, was identified in AT5G22090, which caused a premature stop codon.

To complement the ear1-1 mutant, the complete EAR1 sequence was amplified by PCR using genomic DNA as a template. The resulting DNA fragment was ligated into the pCAMBIA1391 vector at the PstI and BglII restriction enzyme sites. The resulting construct was transformed into Agrobacterium tumefaciens GV3101 and transferred to ear1-1 plants using the floral dip method (Clough and Bent, 1998). The transgenic plants were screened on MS medium containing 30 mg/L hygromycin to obtain homozygous lines for examining ABA phenotypes.

To produce the various truncations to complement the ear1-1 phenotype, the EAR1 promoter was cloned into pCAMBIA1391 between the PstI and EcoRI sites, and the truncated CDS fragments were cloned into the vector at the NcoI and PmlI sites. The constructs were subsequently transformed into Agrobacterium GV3101 and transferred into ear1-1 plants as described above.

Subcellular Localization and GUS Staining

The EAR1 cDNA PCR products were cloned into the XbaI and SpeI sites of a modified vector, pCAMBIA 1300, which contains a superpromoter before the XbaI site and GFP cDNA after the SpeI site. This construct was then transformed into Agrobacterium GV3101 and injected into Nicotiana benthamiana leaves using an HDEL tag (Liu et al., 2011) as an ER marker. The plants were subsequently grown at 18°C under a 12-h-light/12-h-dark cycle in a greenhouse for 48 to 72 h prior to observation. The GFP and RFP signals were detected under a Zeiss LSM 510 META confocal laser-scanning microscope. The GFP images were acquired at excitation of 488 nm and emission of 525 nm, and the RFP images were acquired at excitation of 543 nm and emission at 615 nm.

To determine the subcellular localization of EAR1 in the transgenic plants, full-length EAR1 cDNA was fused with GFP in-frame and cloned into pCAMBIA1391 under the control of the EAR1 native promoter, and the correct construct was transformed into Agrobacterium GV3101. The Agrobacterium was transferred into ear1-1 plants via the floral dip method, and T3 homozygous transgenic seedlings (numbers 6 and 22) that complemented the ABA-sensitive phenotypes of ear1-1 were used for GFP analysis.

For GUS staining, the EAR1 promoter was cloned into pCAMBIA1391 between the PstI and EcoRI sites. The construct was subsequently transformed into Agrobacterium GV3101 and transferred into plants using the floral dip method. T3 hygromycin-resistant transgenic seedlings were used for the GUS staining assay.

Physiological Experiments

All physiological experiments were performed as described previously with some modifications (He et al., 2012). To assay water loss in detached leaves, shoots were cut from plants that had been grown for 4 weeks under normal conditions. The shoots were placed on a piece of weighing paper under a light in the greenhouse, weighed immediately after excision, and periodically weighed every hour. The results are shown as a percentage of the fresh weight. More than two independent experiments were performed, each including three or four replicate shoots per line.

For the stomatal aperture assay, epidermal strips were peeled from 4-week-old plants, and the mesophyll cells were removed with a small brush. To examine ABA-mediated stomatal closure, the epidermal strips were incubated in MES buffer (10 mM MES-KOH, pH 6.15, 10 mM KCl, and 50 μM CaCl₂) in the light for 2 h at 22°C to open the stomata. After the addition of 20 μM ABA to the buffer and incubation of the epidermal strips for an additional 2 h in the light at 22°C, images were obtained under a microscope (Olympus BX53). To examine the ABA-mediated inhibition of stomatal opening in the light, 4-week-old plants were incubated in a dark room for 24 h to close the stomata. Epidermal strips from the plants were then incubated in MES buffer with or without ABA in the light at 22°C for 2 h, after which the epidermal strips were photographed under a microscope (Olympus BX53). The stomata in the images were measured using ImageJ software.

To assess drought-stress tolerance, 7-d-old seedlings were transferred to pots, grown for 2 weeks under normal conditions, and subjected to drought conditions by withholding water from the seedlings for 2 weeks. The drought phenotypes were photographed with a camera (Nikon D80).

Gas-Exchange Measurement

Gas exchange in leaves was measured as described previously (Chen et al., 2011). In brief, the plants were grown at 18°C under a 12-h-light/12-h-dark cycle in a greenhouse for 3 weeks. Water was withheld for 1 week before measurement. The transpiration rate and the photosynthetic rate were determined using an LI-6400XT infrared gas analyzer (LI-COR) at a PAR level of 1000 μmol m⁻² s⁻¹. Each plant in a pot was placed into a leaf chamber, and the transpiration rate and photosynthetic rate were measured after 5 min. For each genotype, plants in three different pots were examined, with similar results.

Thermal Imaging

Thermal imaging of plants was performed as described previously with some modifications (Hua et al., 2012). Briefly, the plants were grown at 18 to 20°C under a 12-h-light/12-h-dark cycle in a greenhouse for 3 weeks. Water was then withheld for 1 week before measurements. Thermal images were obtained using a VarioCAM HD infrared camera (JENOPTIK). Leaf temperature was measured using IRBIS 3 professional software. For each genotype, plants in three different pots were analyzed, with similar results.

Phylogenetic Analysis

The amino acid sequences of EAR1 in each species were acquired from the BLAST tool in the National Center for Biotechnology Information database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple alignments of amino acid sequences were aligned using ClustalX. Phylogenetic analysis was performed with the MEGA4.0 program by the neighbor-joining method with 1000 bootstrap sampling using full-length sequences.

Quantitative RT-PCR

Total RNA was extracted from 7-d-old seedlings with TRIzol reagent (Life Technologies). The RNA was reverse transcribed to cDNA with MMLV reverse transcriptase (Promega). Quantitative RT-PCR was performed on a 7300 Real-Time PCR system (Applied Biosystems) using SYBR Premix Ex Taq (TaKaRa). UBQ was used as an internal control. For each genotype, three different samples were used as three replicates.

Protein Purification

Proteins with different tags were expressed in BL21 Escherichia coli cells with 0.5 mM IPTG for 18 h at 20°C. The culture was then collected by centrifugation at 6000g for 10 min at 4°C.

For His-tagged proteins, the pellet was resuspended in lysis buffer (50 mM NaH₂PO₄, 30 mM NaCl, 10 mM imidazole, pH 8.0, 1 mM PMSF, and 1× cocktail) and sonicated for 10 min. After centrifugation for 30 min at 7000g, the supernatant was incubated with Ni Sepharose (GE) resin for 3 h at 4°C. The resin was washed three times with washing buffer (50 mM NaH₂PO₄, 30 mM NaCl, and 50 mM imidazole, pH 8.0) to remove nonspecifically bound proteins. The His-tagged proteins were eluted from the resin with elution buffer (50 mM NaH₂PO₄, 30 mM NaCl, and 250 mM imidazole, pH 7.4).

For GST-tagged proteins, the pellet was resuspended in 1× PBS (150 mM NaCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 2.7 mM KCl, pH 7.4, 1 mM PMSF, and 1× cocktail) and sonicated for 10 min. After centrifugation for 30 min at 7000g and 4°C, the supernatant was incubated with Glutathione Sepharose 4B (GE) resin for 3 h at 4°C. The resin was washed three times with 1× PBS to remove nonspecifically bound proteins. The GST-tagged proteins were eluted from the resin with GSH buffer (50 mM Tris-HCl, pH 8.0, and 10 mM GSH).

For MBP-tagged proteins, the pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1× cocktail) and sonicated for 10 min. After centrifugation for 30 min at 7000g and 4°C, the supernatant was incubated with amylose resin (NEB) for 3 h at 4°C. The resin was washed three times with lysis buffer to remove nonspecifically bound proteins. The MBP-tagged proteins were eluted from the resin with elution buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, and 10 mM maltose).

The purified proteins were mixed with Quick Start Bradford 1× Dye Reagent (Bio-Rad), and the absorbance was measured at 595 nm. Protein concentrations were calculated based on a standard curve with BSA as a substrate.

Protein Pull-Down Assay

The pull-down assay was performed as described (Ding et al., 2015) with some modifications. In brief, the corresponding CDS of EAR1^121-287, ABI1, or ABI2 was cloned into the pET-30a (+) vector or the pMAL-c5X vector. EAR1^121-287-His protein was purified on Ni Sepharose (GE), and MBP, MBP-ABI1, or MBP-ABI2 was purified on amylose resin (NEB). Purified MBP, MBP-ABI1, or MBP-ABI2 protein (10 μg) was incubated with amylose resin for 2 h in a tube with pull-down binding buffer (150 mM NaCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 2.7 mM KCl, and 0.1% Nonidet P-40, pH 7.4) at 4°C. After a brief centrifugation at 1000g for 3 min at 4°C, the buffer was removed and 2 μg of EAR1^121-287-His protein was added to the resin, along with fresh binding buffer. The tube was rotated at 4°C for 2 h for protein binding. The resin was washed five times with 1× PBS (150 mM NaCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 2.7 mM KCl, pH 7.4) to remove nonspecifically bound protein, combined with 50 μL of 1× PBS and 10 μL of 6× SDS loading buffer, and boiled for 5 min. After centrifugation at 12,000g for 1 min at 22°C, the supernatant was subjected to immunoblotting analysis.

For HAB1, HAB2, AHG1, and AHG3, the corresponding CDS was cloned into pET-28a (+) (for HAB1) or pET-30a (+) (the others) (with His tag), and the proteins were purified on Ni Sepharose (GE). The CDS of EAR1^121-287 was cloned into pGEX-4T-1 (with GST tag), and the proteins were purified on Glutathione Sepharose 4B beads (GE). Subsequently, 10 μg of GST or GST-EAR1 proteins was incubated with Glutathione Sepharose 4B beads for 2 h with pull-down binding buffer at 4°C. The buffer was removed, and 2 μg of HAB1-His, HAB2-His, AHG1-His, or AHG3-His protein was combined with fresh binding buffer and rotated at 4°C for 2 h. The beads were washed five times with washing buffer, combined with 50 μL of 1× PBS and 10 μL of 6×SDS loading buffer, and boiled for 5 min. The supernatant was used for immunoblotting analysis.

Co-IP and LC-MS/MS Assay

The co-IP assay was performed using Arabidopsis protoplasts as described previously (Kong et al., 2015). Briefly, the full-length CDS of EAR1, ABI1, ABI2, HAB1, HAB2, AHG1, and AHG3 were cloned into the pCAMBIA1300 vector and fused to a Myc or Flag tag. The Pro35S:EAR1-Flag constructs were cotransformed into Arabidopsis mesophyll protoplasts along with Pro35S:ABI1-Myc, Pro35S:ABI2-Myc, Pro35S:HAB1-Myc, Pro35S:HAB2-Myc, Pro35S:AHG1-Myc, and Pro35S:AHG3-Myc. After incubation for 14 to 16 h, total proteins were extracted from the protoplasts, and the extracts were incubated with anti-c-Myc-Agarose Affinity Gel (Sigma-Aldrich) at 4°C for 3 h. The beads were washed five times with PBS (pH 7.5) and boiled for 5 min in 6× SDS loading buffer. After centrifugation at 12,000g for 1 min, the supernatants were used for immunoblotting analysis.

Transgenic plants harboring Pro35S:EAR1-Flag (OE-16^#) were also subjected to co-IP assays as described previously with some modifications (Gao et al., 2015). Briefly, total proteins were extracted from the wild type and OE-16^# with immunoprecipitation buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.6, 2 mM EDTA, 0.5% Nonidet P-40, and 1× protease inhibitor [Roche]) and incubated with anti-Flag M2 Affinity gel (Sigma-Aldrich) at 4°C for 3 h. The beads were subsequently washed five times with PBS (pH 7.5) at 4°C for 10 min each time. After centrifugation at 1000g for 3 min, the beads were boiled for 5 min in 100 μL of PBS and 20 μL of 6× SDS loading buffer. The immunoprecipitated proteins were used for immunoblotting analysis, in which the ABI1 and ABI2 proteins were detected using anti-ABI1 and anti-ABI2 antibodies, respectively, and the EAR1 proteins were detected using an anti-Flag antibody (Sigma-Aldrich, F1804) as a loading control. The ABI2 polyclonal antibodies were produced by Beijing Protein Innovation. Briefly, a BglII/EcoRI fragment containing the full-length ABI2 open reading frame was cloned into the BamHI/EcoRI sites of the pET-28a vector to express 6× His-tagged ABI2 protein. The fusion protein was expressed in E. coli, purified, and used as an antigen to immunize rabbits for the production of polyclonal antiserum. Antigen affinity-purified anti-ABI2 antibodies were used in immunoblot analysis. The specificity of the ABI2 antibodies was checked by comparing the abi2-2 mutant with the wild type (Supplemental Figure 4). The immunoprecipitated proteins were also used for LC-MS/MS analysis on a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Scientific) as described previously (Kong et al., 2015).

BiFC Assay

BiFC assays were performed as described previously (Waadt and Kudla, 2008). Briefly, the full-length CDS of EAR1 was cloned into the pSPYCE(M) vector to generate the EAR1-cYFP plasmid, and ABI1, ABI2, HAB11, HAB2, AHG1, and AHG3 were cloned into the pSPYNE173 vector to construct the ABI1-nYFP, ABI2-nYFP, HAB1-nYFP, HAB2-nYFP, AHG1-nYFP, and AHG3-nYFP plasmids, respectively. To produce truncated EAR1 and ABI1, the corresponding CDS of EAR1 and ABI1 were amplified and cloned into the pSPYCE(M) and pSPYNE173 vectors, forming the EAR1^1-287-cYFP, EAR1^1-120-cYFP, EAR1^121-287-cYFP, EAR1^191-287-cYFP, EAR1^191-270-cYFP, EAR1^288-463-cYFP, ABI1^1-120-nYFP, ABI1^1-130-nYFP, and ABI1^131-434-nYFP constructs. The plasmids were then transformed into Agrobacterium GV3101 cells, and different combinations were mixed for injection into Nicotiana benthamiana leaves with the silencing suppressor P19 strain. The plants were grown at 18°C under a 12-h-light/12-h-dark cycle in a greenhouse for 48 to 72 h, and YFP signals were observed under a confocal laser-scanning microscope (Zeiss LSM 510 META).

Yeast Three-Hybrid Assay

The yeast three-hybrid assay was performed as described previously (Ding et al., 2015). In brief, the plasmids (pGADT7-ABI1 and pBridge-PYR1 or pGADT7-ABI1 and pBridge-PYR1-EAR1) were cotransformed into yeast strain AH109. Positive clones were selected on synthetic complete medium lacking Leu, Trp, Ade, and Met. β-Galactosidase activity was measured using a Yeast β-Galactosidase Assay Kit (product 75768, Thermo Scientific) following the manufacturer’s instructions. Ten positive clones were used for the β-galactosidase activity assay in each experiment. Three independent experiments were performed, with similar results.

Cell Fractionation Assay

Cell fractionation assays were performed as described previously (Wang et al., 2011b). Briefly, 0.5-g samples of 2-week-old OE-16^# transgenic plants were ground in liquid nitrogen and mixed with 2 mL of fractionation lysis buffer (20 mM Tris-HCl, pH 7.5, 25% glycerol, 2 mM EDTA, 2.5 mM MgCl₂, 20 mM KCl, 250 mM sucrose, with the addition of 5 mM DTT with 1× protease inhibitor cocktail [Roche] before use). The mixture was vortexed and filtered through a double layer of Miracloth (Millipore). The flow-through was centrifuged at 1500g for 10 min at 4°C, after which the supernatant was centrifuged at 10,000g for 10 min at 4°C, and the pellet was collected as the cytoplasmic fraction. The pellet from the first centrifugation was washed five times or more with NRBT buffer (20 mM Tris-HCl, pH 7.5, 25% glycerol, 2.5 mM MgCl₂, and 0.2% Triton X-100) until it turned oyster white. The pellet was resuspended in 400 μL of NRB2 (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM MgCl₂, 0.5% Triton X-100, and 5 mM β-mercaptoethanol supplemented with 1× protease inhibitor cocktail) and added on top of a layer of 400 μL of NRB3 (20 mM Tris-HCl, pH 7.5, 1.7 M sucrose, 10 mM MgCl₂, 0.5% Triton X-100, and 5 mM β-mercaptoethanol supplemented with 1× protease inhibitor cocktail). The suspension was centrifuged at 16,000g for 45 min at 4°C, and the pellet was collected as the nuclear fraction. Both the cytoplasmic and nuclear fractions were resuspended in 2× SDS loading buffer and boiled for 5 min for the immunoblot assays. EAR1 proteins were detected using an anti-Flag antibody (Sigma-Aldrich, F1804). Tubulin was detected with an anti-tubulin antibody (Sigma-Aldrich, T3526) as a cytoplasmic marker (Saslowsky et al., 2005). H3 proteins were detected using an anti-H3 antibody (Millipore, 17-10046) as a nuclear marker (Feys et al., 2005).

The microsomal fractionation assay was performed as described previously (Bueso et al., 2014). In brief, 0.1-g samples of 2-week-old OE-16^# transgenic plants were ground in liquid nitrogen and mixed with 800 μL of LE buffer (80 mM Tris-HCl, pH 7.5, 12% sucrose, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 5 mg/mL leupeptin). The crude extract was centrifuged at 10,000g for 10 min at 4°C, and the supernatant was collected as total protein. The supernatant was centrifuged at 100,000g for 1 h, and the new supernatant was collected as the soluble fraction; the pellet was resuspended in LE buffer and collected as the microsomal fraction. Both fractions were analyzed by immunoblotting, and EAR1 proteins were detected using an anti-Flag antibody (Sigma-Aldrich).

Measurement of PP2C Activity

PP2C activity was measured using a Ser/Thr Phosphatase Assay Kit (Promega) as described previously (Yu et al., 2012). In brief, the corresponding CDS of ABI1, ABI2, HAB1, HAB2, AHG1, AHG3, EAR1^121-287, EAR1^191-287, EAR1^121-210, EAR1^201-287, EAR1^191-260, and EAR1^191-270 were fused with the pET-28a (+) (for HAB1) or pET-30a (+) (the others) vectors, and the constructs were transformed into the E. coli strain BL21. The recombinant proteins were purified on Ni Sepharose (GE) and eluted with elution buffer (50 mM Tris-HCl, pH 7.2, and 250 mM imidazole). None of the buffers used for protein purification contained phosphate. To measure phosphatase activity, different combinations of EAR1 truncated proteins and PP2C proteins were mixed with 5 μL of 1 mM phosphopeptide [RRA(phosphoT)VA] and 10 μL of PP2C 5× reaction buffer (250 mM imidazole, pH 7.2, 1 mM EGTA, 25 mM MgCl₂, 0.1% β-mercaptoethanol, and 0.5 mg/mL BSA) at 25°C for 15 min. The reactions were stopped by adding 50 μL of molybdate dye/additive mixture, and the plate was incubated at room temperature for 15 min. The absorbance was measured at 630 nm in a Microplate Reader (Power Wave XS2). As EAR1^121-210, EAR1^201-287, EAR1^191-260, and EAR1^191-270 did not enhance PP2C activity, they were considered to serve as controls.

To measure PP2C activity in lysates, total proteins (0.1 g) from the wild type, ear1-1, OE-16^#, OE-28^#, 3m, and 4m were extracted in phosphatase storage buffer (20 mM Tris-HCl, pH 7.5, 20 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 mM DTT, 0.5% Triton X-100, and 50% glycerol) with 1× protease inhibitor cocktail (Roche), and the homogenized lysate was centrifuged at 100,000g for 60 min at 4°C. The supernatant was passed through Sephadex G-25 resin to remove the endogenous phosphates in the tissue extract, and the filtered lysate was used to measure PP2C activity as described above but with the addition of 5 µM okadaic acid.

In Vitro Phosphorylation Assay

The in vitro phosphorylation assay was performed as described previously (Ding et al., 2015). In brief, the corresponding CDS of OST1, ABI1, and EAR1^121-287 were fused with the pET-30a (+) vector (Novagen). The correct plasmids were transformed into E. coil BL21 (DE3) cells, and the recombinant proteins were purified on Ni Sepharose (GE). The in vitro phosphorylation assay was performed using 4 μg of OST1-His with different combinations of ABI1-His and EAR1^121-287-His recombinant proteins. The mixed proteins were incubated in kinase buffer (100 mM HEPES-NaOH, pH 7.5, 10 mM MgCl₂, 2 mM DTT, and 0.5 mM ATP) with 2 μCi of [γ-³²P]ATP in a 20-μL volume at 30°C for 20 min. The reactions were stopped by adding 4 μL of 6× SDS loading buffer, and the proteins were separated by 10% SDS-PAGE. The gel was washed three times with distilled water, and radioactivity in the autoradiograph was detected using X-OMAT BT Film (Carestream) or a Typhoon 9410 imager.

In-Gel Kinase Assay

The in-gel kinase assay was performed as described previously (Kong et al., 2015). In brief, total proteins were extracted from the wild type, OE-16^#, ear1, and ost1 with lysis buffer (50 mM HEPES-KOH, pH 7.5, 5 mM EDTA, 5 mM EGTA, 2 mM DTT, 25 mM NaF, 1 mM Na₃VO₄, 20% glycerol, and 1× protease inhibitor cocktail [Roche]). Total proteins (80 μg) were separated on an SDS-PAGE gel containing 0.1 mg/mL GST-Δ(73-118) substrate and washed three times with SDS removing buffer (25 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 5 mM NaF, 0.1 mM Na₃VO₄, 0.5 mg/mL BSA, and 0.1% Triton X-100) at room temperature for 20 min each time. The gel was then washed three times with renaturing buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 5 mM NaF, and 0.1 mM Na₃VO₄) for 1, 12, and 1 h at 4°C to renature the proteins in the gel. After incubation in kinase reaction buffer (40 mM HEPES-KOH, pH 7.5, 1 mM DTT, 2 mM EGTA, 12 mM MgCl₂, and 0.1 mM Na₃VO₄) for 30 min, the gel was incubated in 30 mL of fresh kinase reaction buffer with 60 μCi of [γ-³²P]ATP and 9 μL of 1 mM cold ATP at room temperature for 3 h. Finally, the gel was washed five times with 5% trichloroacetic acid and 1% sodium pyrophosphate for 30 min each time. The gel was covered with a storage phosphor screen (GE), and radioactivity was detected with a Typhoon 9410 imager.

The primers used in this study are listed in the Supplemental Data Set 3.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: EAR1, At5g22090; ABI1, At4g26080; ABI2, At5g57050; HAB1, At1g72770; HAB2, At1g17550; AHG1, At5g51760; AHG3, At3g11410; HAI1, At5g59220; HAI2, At1g07430; HAI3, At2g29380; SnRK2.6/OST1, At4g33950; RD29A, At5g52310; RD29B, At5g52300; RAB18, At1g43890; ABF4, At3g19290; P5CS1, At2g39800; and KIN1, At1g14370. Mutants used in this article can be obtained from the ABRC under the following accession numbers: ear1-2 (SALK_057606), abi1-2 (SALK_072009), abi2-2 (SALK_015166), hab1-1 (SALK_002104), ost1/snrk2.6 (SALK_008068), snrk2.2 (GABI-Kat 807G04), and snrk2.3 (SALK_107315).

Supplemental Data

• Supplemental Figure 1. Phenotypes of SALK_057606 (ear1-2) and ear1-c Mutants.

• Supplemental Figure 2. Phylogenetic Analysis of EAR1 Sequences.

• Supplemental Figure 3. Comparison of Plant Phenotypes among the Wild Type, ear1-1, and EAR1-Overexpressing Lines OE-16^# and OE-28^#.

• Supplemental Figure 4. EAR1 Interacts with Clade A PP2Cs.

• Supplemental Figure 5. EAR1 Enhances the Activity of the Clade A PP2Cs.

• Supplemental Figure 6. EAR1 Does Not Influence the Interaction between ABI1 and PYR1 or the Degradation of ABI1.

• Supplemental Figure 7. Protein Characteristics of EAR1 and PP2Cs.

• Supplemental Figure 8. EAR1 Negatively Regulates the Expression of ABA-Inducible Genes.

• Supplemental Figure 9. Subcellular Localization of EAR1.

• Supplemental Data Set 1. Alignments Use to Generate the Phylogeny Presented in Supplemental Figure 2A.

• Supplemental Data Set 2. LC-MS/MS Results for EAR1 Interaction Proteins without ABA Treatment in Arabidopsis.

• Supplemental Data Set 3. LC-MS/MS Results for EAR1 Interaction Proteins with ABA Treatment in Arabidopsis.

• Supplemental Data Set 4. Primers Used in This Study.