Selective inhibition of clade A phosphatases type 2C by PYR/PYL/RCAR abscisic acid receptors

Abstract

Clade A protein phosphatases type 2C (PP2Cs) are negative regulators of abscisic acid (ABA) signaling that are inhibited in an ABA-dependent manner by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) intracellular receptors. We provide genetic evidence that a previously uncharacterized member of this PP2C family in Arabidopsis (Arabidopsis thaliana), At5g59220, is a negative regulator of osmotic stress and ABA signaling and that this function was only apparent when double loss-of-function mutants with pp2ca-1/ahg3 were generated. At5g59220-green fluorescent protein and its close relative PP2CA-green fluorescent protein showed a predominant nuclear localization; however, hemagglutinin-tagged versions were also localized to cytosol and microsomal pellets. At5g59220 was selectively inhibited by some PYR/PYL ABA receptors, and close relatives of this PP2C, such as PP2CA/ABA-HYPERSENSITIVE GERMINATION3 (AHG3) and AHG1, showed a contrasting sensitivity to PYR/PYL inhibition. Interestingly, AHG1 was resistant to inhibition by the PYR/PYL receptors tested, which suggests that this seed-specific phosphatase is still able to regulate ABA signaling in the presence of ABA and PYR/PYL receptors and therefore to control the highly active ABA signaling pathway that operates during seed development. Moreover, the differential sensitivity of the phosphatases At5g59220 and PP2CA to inhibition by ABA receptors reveals a functional specialization of PYR/PYL ABA receptors to preferentially inhibit certain PP2Cs.

Figure 1.

A, Schematic diagram of the At5g59220 gene showing the position of the T-DNA insertion in the hai1-1 mutant. RT-PCR analysis of mRNAs from wild-type and hai1-1 mutant seedlings is shown. Primers FMSTV2CB and R2CB1426 were used to amplify part of the At5g59220 cDNA. LB, Left border; RB, right border. B, Seedling establishment of the Columbia (Col) wild type, hai1-1, pp2ca-1, and the double mutant in medium supplemented with ABA, mannitol, or Glc. Data show the percentage of seeds that germinated and developed green cotyledons in the different media at 5 d. Values are averages ± se for three independent experiments (200 seeds each). * P < 0.01 (Student’s t test) with respect to the wild type. C, Photograph of a representative experiment taken 10 d after sowing, with a magnification of representative seedlings grown on MS plates supplemented with 0.2 m mannitol. D, Seedling establishment of wild-type, 35S:HA-PP2CA, and 35S:HA-At5g59220 lines in medium supplemented with 1 μm ABA (top panel), 0.2 m Glc, or 0.2 m mannitol (bottom panel). Approximately 200 seeds of each genotype were sown on each plate and scored 4 d later. Photographs were taken after 8 d. OE indicates overexpression lines. [See online article for color version of this figure.]

Figure 2.

A, ABA-hypersensitive growth inhibition of pp2ca-1 hai1-1 and hab1-1 abi1-2 double mutants compared with the wild type and single parental mutants. Photographs show representative seedlings 10 d after the transfer of 4-d-old seedlings from MS medium to plates lacking or supplemented with 10 μm ABA. B, Quantification of ABA-mediated root growth inhibition of pp2ca-1 hai1-1 and hab1-1 abi1-2 double mutants compared with the wild type and single parental mutants. Data are averages ± se from three independent experiments (n = 15 each). * P < 0.01 (Student’s t test) with respect to the wild type. C, Relative expression of ABA-responsive genes in the pp2ca-1 hai1-1 double mutant compared with the wild type and single parental mutants. RT-qPCR analyses were performed in triplicate on RNA samples of 2-week-old seedlings that were either mock treated or 10 μm ABA treated for 3 h. Numbers indicate the expression levels of the genes in each mutant genotype with respect to the wild type in samples treated with ABA (value of 1). Expression of RD29A, KIN1, RAB18, and RD29B was up-regulated 6-, 312-, 86-, and 634-fold by ABA treatment in the wild type, respectively. D, Reduced water loss of pp2ca-1 hai1-1 compared with the Columbia (Col) wild type. Five leaves at the same developmental stage were detached from 21-d-old plants, and fresh weight was determined after submitting them to the drying atmosphere of a flow laminar hood (n = 4 plants per experiment). * P < 0.05 (Student’s t test) with respect to the wild type. [See online article for color version of this figure.]

Figure 3.

Subcellular localization of PP2CA and At5g59220. A, Subcellular localization of PP2CA-GFP and At5g59220-GFP proteins transiently expressed in tobacco cells. Epifluorescence and bright-field images of epidermal leaf cells infiltrated with a mixture of Agrobacterium suspensions harboring the indicated constructs and the silencing suppressor p19 are shown. SWI3B is a nuclear protein that forms part of the SWI/SNF chromatin-remodeling complexes (Saez et al., 2008). The N-terminal extension (residues 1–97) and the catalytic core (residues 98–413) of At5g59220 were expressed as fusions with GFP. B, Biochemical fractionation of HA-PP2CA and HA-At5g59220 proteins. Plant material was obtained from epitope HA-tagged PP2CA or At5g59220 transgenic lines after mock treatment or 50 μm ABA treatment for 1 h. Samples were analyzed using anti-HA, anti-histone 3 (α-H3), anti-plasma membrane H⁺-ATPase antibodies and Ponceau staining of Rubisco. Localization of HA-PP2CA and HA-At5g59220 proteins in soluble (S), total nuclear (N), nuclear soluble (Ns), nuclear insoluble (Ni), cytosolic (C), and microsomal (M) fractions is indicated. Histograms show the relative amounts of each protein in the different fractions. OE indicates overexpression lines. [See online article for color version of this figure.]

Figure 4.

Differential sensitivity of PP2CA, At5g59220, and AHG1 to ABA-dependent PYR/PYL-mediated inhibition. A, Phosphatase activity of the different PP2Cs was measured in vitro using a phosphopeptide substrate in the absence or presence of 0.5, 1, 5, 10, 20, 40, or 50 μm ABA and the indicated receptors. For the sake of clarity, only data for 10 and 50 μm ABA are shown as well as IC₅₀ values (n.d., not determined at IC₅₀ > 50 μm). Data are averages ± sd for three independent experiments. Phosphatase assays were performed in a 100-μL reaction volume containing 2.3 μg of His₆-PP2CA, 2.1 μg of His₆-ΔNAt5g59220, or 2.5 μg of His₆-AHG1 and between 5 and 5.7 μg of the different His₆-PYR/PYL proteins in order to obtain a 1:4 phosphatase:receptor stoichiometry. The activities of the PP2C recombinant proteins in the absence of ABA (100% activity) were 12.2 ± 0.3, 13.3 ± 0.2, and 11.0 ± 0.2 nmol inorganic phosphate min⁻¹ mg⁻¹, respectively. In order to check the effect of the HIS elution buffer on the PP2C activity, we performed an assay lacking PYR/PYL proteins but adding an equivalent volume of HIS elution buffer. B, In vitro OST1 kinase activity in the absence or presence of 30 μm ABA and the indicated receptors. A 1:10 phosphatase:receptor stoichiometry was used in this assay. The quantification of autoradiography (numbers below) shows the percentage of OST1 phosphorylation in each reaction relative to the first reaction (100%; phosphorylation of OST1 in the absence of PP2Cs). C, Dephosphorylation of ΔCABF2 by PP2CA and At5g59220 in the absence or presence of 30 μm ABA and PYL8.