PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis

Abstract

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1-PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1-ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling.

Figure 1

Constitutive expression of YFP–ABI1 causes ABA insensitivity. (a) Western blot analysis of YFP-tagged ABI1 (left) and YFP control (right) proteins in transgenic Arabidopsis. Stars indicate predicted YFP–ABI1 and YFP bands. Arrow head: non-specific band. (b) Morphology and subcellular localization of Arabidopsis plants constitutively expressing YFP–ABI1 (left) and YFP (right) at the rosette plant stage. Plants were grown for 5 weeks in soil. (c) ABA-insensitive phenotype of constitutively expressed YFP–ABI1 (left) and ABA response in control YFP-expressing seedlings (right). Top: in the presence of 1 μm ABA. Bottom: without added ABA. Transgenic plant seeds were sown on agar MS plates with or without 1 μm exogenous ABA. (d) ABA-dependent root growth responses of Arabidopsis seedlings constitutively expressing YFP and YFP–ABI1. Seedlings were germinated and grown on hormone-free MS plates for 5 days and then transferred to MS plates containing the indicated ABA concentrations. Root length was measured 4 days after transfer. Error bars show standard deviations. (e) Time course experiments of ABA-induced stomatal closing in YFP control and YFP–ABI1 expressing leaves (genotype blind experiments). Stomatal apertures were individually mapped and images captured (Siegel et al., 2009) and measured before and after addition of 1 μm ABA. Average stomatal apertures at time −15 min: 3.47 ± 0.14 μm (YFP plants), 3.75 ± 0.18 μm (YFP–ABI1 plants). YFP plants: n = 23 stomata, YFP–ABI1 plants: n = 26 stomata. Error bars show SEM.

Figure 2

ABA causes ABI1–PYR1 interaction in Arabidopsis. (a) Example of raw tandem mass spectrometry data identifying PYR1 peptide in YFP–ABI affinity purified samples. The predicted PYR1 peptide sequence is shown in the top inset. (b) Increasing concentrations of exogenously applied ABA strongly enhanced co-immunoprecipitation of YFP–ABI1 with PYR1 in Arabidopsis. Transgenic Arabidopsis plants were exposed to ABA for 3 h. (c) ABA triggers interaction of YFP–ABI1 with PYR1 within 5 min of exposure of Arabidopsis plants to exogenous ABA. Left: Total protein extracts (left, Input) from YFP–ABI1 plants. Plants were grown on MS plates for 3 weeks and were treated with or without 100 μm exogenous ABA. After co-immunoprecipitation using anti-GFP beads, input and co-immunoprecipitated samples were detected with anti-PYR1 and anti-GFP antibodies.

Figure 3

ABI1 PP2C co-immunoprecipitates with all three ABA signalling SnRK2 kinases in plants. YFP–ABI1 co-immunoprecipitates with HA–SnRK2.2, HA–SnRK2.3 and HA– OST1/SnRK2.6 both in the presence and absence of exogenously applied ABA. Total protein extracts (left, Input) from transformed Nicotiana benthamiana leaves were harvested 3 days after inoculation and were treated with or without 100 μm ABA for 24 h before harvesting. After co-immunoprecipitation using an anti-HA matrix, input and immunoprecipitated samples were detected with anti-GFP and anti-HA antibodies.

Figure 4

Interaction of PYR1 with SnRK2.3 and OST1. YFP–PYR1 co-immunoprecipitates with HA–SnRK2.3. In contrast HA–OST1/SnRK2.6 did not co-immunoprecipitate with YFP–PYR1. Total protein extracts (left, Input) from transformed Nicotiana benthamiana leaves were harvested 3 days after inoculation and were treated with or without 100 μm ABA for 24 h before harvesting. After co-immunoprecipitation using an anti-HA matrix, input and immunoprecipitated samples were detected with anti-GFP and anti-HA antibodies.

Figure 5

ABA-induced stomatal closure and ABA-inhibition of stomatal opening but not Ca²⁺ -induced stomatal closure are impaired in pyr1pyl1pyl2pyl4 quadruple mutant plants. (a) ABA-induced stomatal closure in pyr1pyl1pyl2pyl4 quadruple mutant and wild type rosette leaves treated with the indicated ABA concentrations for 1 h (n = 4 experiments, 30 stomata per experiment and condition; genotype and [ABA] blind experiments). (b) ABA inhibition of stomatal opening in pyr1pyl1pyl2pyl4 quadruple mutant and wild type abaxial leaf epidermes treated with the indicated ABA concentrations for 1 h (n = 3 experiments, 30 stomata per experiment and condition; genotype and [ABA] blind experiments). Closed means incubation in the dark for 3 h (before light and ABA treatment). (c) Stomatal closure in response to four repetitive extracellular Ca²⁺ pulses (5 min each) is not impaired in pyr1pyl1pyl2pyl4 quadruple mutant suggesting that PYR/PYL/RCARs function upstream of Ca²⁺. Four 5-min extracellular applications of 1 mm CaCl₂ and 1 mm KCl were sequentially applied to abaxial leaf epidermes (black bars at top) followed by 50 mm KCl and 0 added CaCl₂ exposure (White bars at top) (n = 31 individually mapped stomata for both wild type and pyr1pyl1pyl2pyl4 quadruple mutant) Average stomatal apertures at time = −15 min: 5.24 ± 0.19 μm (wild type), 5.52 ± 0.14 μm (pyr1pyl1pyl2pyl4 quadruple mutant). Error bars show SEM.