The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA

Abstract

To identify new loci in abscisic acid (ABA) signaling, we screened a library of 35ScDNA Arabidopsis (Arabidopsis thaliana)-expressing lines for ABA-insensitive mutants in seed germination assays. One of the identified mutants germinated on 2.5 microm ABA, a concentration that completely inhibits wild-type seed germination. Backcrosses and F2 analyses indicated that the mutant exhibits a dominant phenotype and that the ABA insensitivity was linked to a single T-DNA insertion containing a 35ScDNA fusion. The inserted cDNA corresponds to a full-length cDNA of the AtPP2CA gene, encoding a protein phosphatase type 2C (PP2C). Northern-blot analyses demonstrated that the AtPP2CA transcript is indeed overexpressed in the mutant (named PP2CAox). Two independent homozygous T-DNA insertion lines, pp2ca-1 and pp2ca-2, were recovered from the Arabidopsis Biological Resource Center and shown to lack full-length AtPP2CA expression. A detailed characterization of PP2CAox and the T-DNA disruption mutants demonstrated that, whereas ectopic expression of a 35SAtPP2CA fusion caused ABA insensitivity in seed germination and ABA-induced stomatal closure responses, disruption mutants displayed the opposite phenotype, namely, strong ABA hypersensitivity. Thus our data demonstrate that the PP2CA protein phosphatase is a strong negative regulator of ABA signal transduction. Furthermore, it has been previously shown that the AtPP2CA transcript is down-regulated in the ABA-hypersensitive nuclear mRNA cap-binding protein mutant abh1. We show here that down-regulation of AtPP2CA in abh1 is not due to impaired RNA splicing of AtPP2CA pre-mRNA. Moreover, expression of a 35SAtPP2CA cDNA fusion in abh1 partially suppresses abh1 hypersensitivity, and the data further suggest that additional mechanisms contribute to ABA hypersensitivity of abh1.

Figure 1.

The ABA-insensitive 393.1 mutant is an overexpressor of AtPP2CA encoding a PP2C. A, Schematic representation of the 393.1 mutant T-DNA containing the full-length cDNA of AtPP2CA. The AtPP2CA cDNA is inserted at the original XhoI and NotI restriction sites of the 35SpBARN vector. Arrows indicate the direction of transcription. 35S-F and NOS-R are the primers used for amplifying the cDNA. BARN-LB1 is the primer used for T-DNA left-border sequencing. Note that the XhoI restriction site no longer exists because it has been filled with T4 polymerase during the original cloning procedure (LeClere and Bartel, 2001). B, Northern-blot analysis of AtPP2CA in wild-type, abh1, pp2ca-1, and PP2CAox leaves. Hybridization signals with ACTIN7 cDNA (ACT7) were used for standardization of RNA and the value obtained from wild-type leaves was set to 1.

Figure 2.

Constitutive expression of AtPP2CA results in ABA insensitivity, while disruption of AtPP2CA causes ABA hypersensitivity during seed germination. A, Comparison of germination rates of wild-type (Col-0; black circles), pp2ca-1 (white triangles), abh1 (black squares), and PP2CAox (white circles) seeds exposed to 0, 0.3, 0.5, 1, and 2.5 μm ABA at 5 d. Data represent the mean ± sem of three independent experiments with 36 seeds per genotype and experiment. See Supplemental Figure 1A for similar data with pp2ca-2. Error bars are smaller than symbols, if not visible. B, Comparison of germination for wild-type, pp2ca-1, abh1, and PP2CAox seeds at 1 μm ABA or in the absence of ABA after 5 d. C, Germination of seeds from wild-type and 14 individual T1 lines with constitutive 35S∷AtPP2CA expression at 2.5 μm ABA 5 d after germination.

Figure 3.

Disruption of AtPP2CA causes reduced root elongation in response to ABA. A, Schematic representation of the genomic organization of the AtPP2CA gene with four exons (black boxes). Positions of the pp2ca-1 and pp2ca-2 T-DNA insertions are indicated and orientation of the left-border sequence of the respective T-DNAs is represented by broken arrows. B, RT-PCR analysis shows no full-length transcript in AtPP2CA T-DNA disruption lines pp2ca-1 and pp2ca-2. PCR reactions were performed with oligonucleotides PP2CAEx1-F and PP2CAEx4-R (Table I) and samples were withdrawn from the reaction after 28, 32, and 36 cycles. Amplification of EF1α cDNA with primers EF1α-F and EF1α-R (Table I) was used for controls. C, Comparison of root elongation of wild-type (white bars), pp2ca-1 (dark gray bars), pp2ca-2 (light gray bars), and PP2CAox (black bars) seedlings; 6-d-old seedlings were transferred to plates supplemented with 0, 2.5, 5, 10, 25, and 50 μm ABA, and root elongation was monitored after 6 d. Each data point represents the mean of three independent experiments with eight seedlings each. Asterisks (*) indicate a significant change between wild-type and pp2ca-1 or pp2ca-2 plants (P < 0.001). D, Growth of wild type, pp2ca-1, and PP2CAox on 0.25× Murashige and Skoog medium supplemented with 50 μm ABA. The photographs show plants on plates with 0 (top) and 50 μm ABA (bottom) 18 d after transfer of 6-d-old seedlings from 0.25× Murashige and Skoog medium.

Figure 4.

AtPP2CA transcripts are rapidly and highly up-regulated by both ABA and drought treatments. Northern-blot analyses of AtPP2CA in wild-type leaves either treated with ABA (50 μm; left) or excised and subjected to desiccation (right). Total RNA was extracted from leaves at times specified by the number above each lane. Hybridization signals with ACTIN7 cDNA (ACT7) were used for standardization of equal amounts of RNA. Values obtained prior to the indicated treatments were set to 1.

Figure 5.

AtPP2CA modulates the stomatal response to ABA. A, Ectopic expression of AtPP2CA in plants causes enhanced leaf evaporation rate compared to wild type. Loss of fresh weight of detached rosette leaves at the same developmental stages was measured for wild-type (black circles), pp2ca-1 (white triangles), and PP2CAox plants (white circles) at the indicated time points. Data represent the mean of three independent experiments ± sem. B, Stomatal closing is ABA hypersensitive in pp2ca-1 and ABA insensitive in PP2CAox plants. Stomatal aperture measurements of wild type (white bars), pp2ca-1 (shaded bars), and PP2CAox (black bars) in response to 0, 1, and 10 μm ABA. Data represent the mean of n = 4 independent experiments ± sem with 4 × 50 stomata per data point. Asterisks (*) indicate significant changes between the indicated genotype and wild type (P < 0.001). C, Stomatal closing is ABA hypersensitive in pp2ca-2. Stomatal aperture measurements of wild type (white bars) and pp2ca-2 (shaded bars) in response to 0, 1, and 10 μm ABA. Data represent the mean of two independent experiments ± sem with 2 × 50 stomata per data point. Asterisks (*) indicate significant changes between the indicated genotype and wild type (P < 0.001). See Supplemental Figure 1, B and C, for stomatal aperture ratios from experiments in Figure 5, B and C.

Figure 6.

Analysis of AtPP2CA intron splicing in wild type and abh1. A, Schematic representation of AtPP2CA genomic organization with exons (black boxes) and introns (lines between exons). Positions of primers (arrows) used for the RT-PCR analysis are indicated and resulting amplification products (dotted lines) are shown for each PCR reaction performed, numbered from 1 to 4. Product sizes for full-length amplification, corresponding to completely unspliced transcripts, or for smaller fragments, corresponding to fully or partially processed mRNAs, are given for each PCR reaction. B, DNA fragments of fully and partially spliced AtPP2CA mRNA amplification products (36 cycles) generated in PCR reactions 1 to 4 (Fig. 6A) for wild-type controls (C) and abh1 (a) are size fractionated on a 1.8% agarose gel. Amplification (28 cycles) of the EF1α was used as internal control. Sizes of coelectrophoresed DNA standard fragments are given in kilobase pairs.

Figure 7.

Ectopic expression of AtPP2CA partially suppresses the ABA hypersensitivity of abh1. A, Comparison of germination rates of wild type (black circles), PP2CAox in wild type (white circles), abh1 (black squares), and seeds from three independent abh1 35S∷AtPP2CA (white diamonds) lines germinated on Murashige and Skoog plates containing 0, 0.3, 0.5, 1, and 2.5 μm ABA after 5 d. Data represent the mean ± sem of three independent experiments. Error bars are smaller than symbols, if not visible. Data for wild type, abh1, and PP2CAox in A are the same as in Figure 2A for reference purposes. B, RT-PCR analysis of AtPP2CA transcript levels in wild type, abh1, and the three independent abh1 35S∷AtPP2CA lines displayed in A from the more sensitive to the more ABA resistant. DNA fragments from RT-PCR analyses of EF1α were used for standardization of equal amplification rates and the value obtained for AtPP2CA quantity in abh1 was set to 1. RT-PCR reactions were performed for 20, 24, 28, and 32 cycles to quantify relative mRNA levels and representative images after 28 cycles are shown.