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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Plant J. 2009 Oct 26;61(2):290–299. doi: 10.1111/j.1365-313X.2009.04054.x

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

Noriyuki Nishimura 1, Ali Sarkeshik 2, Kazumasa Nito 3, Sang-Youl Park 4, Angela Wang 1, Paulo Carvalho 2, Stephen Lee 1, Daniel F Caddell 1, Sean R Cutler 4, Joanne Chory 3, John R Yates III 2, Julian I Schroeder 1,5
PMCID: PMC2807913  NIHMSID: NIHMS152710  PMID: 19874541

Summary

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 signaling, to identify redundant and early ABA signaling 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 signaling components were isolated including SnRK2 protein-kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signaling 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 analyzed 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 signaling.

Keywords: Abscisic acid, ABI1, ABI1 interacting proteins, proteome, PYR/PYL/RCAR

Introduction

The plant hormone abscisic acid (ABA) controls important abiotic stress-induced and developmental responses, including maintenance of seed dormancy, seed development, growth regulation and stomatal closure. Forward and reverse genetic analyses have led to the identification of many loci encoding ABA signaling components that function in ABA metabolism and in ABA signaling (Finkelstein et al. 2002; Nambara and Marion-Poll 2005; Israelsson et al. 2006; Yamaguchi-Shinozaki and Shinozaki 2006). However, genetic redundancy has hampered the identification of many important ABA signaling components. Furthermore, proteins that mediate ABA reception are a subject of present debate and insufficient evidence exists on the interactions of some of the candidate proteins with known ABA signaling components (McCourt and Creelman 2008).

Protein phosphorylation and dephosphorylation events are important mediators of ABA signal transduction. ABA-activated SNF1-related protein kinases 2 (SnRK2s) and Ca2+ dependent protein kinases (CDPKs) are positive transducers of ABA signaling (Li et al. 2000; Mustilli et al. 2002; Yoshida et al. 2002; Mori et al. 2006; Fujii et al. 2007; Zhu et al. 2007). Furthermore calcineurin B-like proteins (CBLs) and CBL-binding protein kinases (CIPKs), encode another class of SNF1-related protein kinases that negatively affect ABA responses (Hrabak et al. 2003; Batistic and Kudla 2009; Luan 2009). The ABA-activated protein kinase OST1 (SnRK2.6/SnRK2E) functions as a positive regulator of ABA-induced stomatal closure (Mustilli, et al. 2002; Yoshida, et al. 2002). SnRK2.2 and SnRK2.3 are homologs of OST1, and are also activated by ABA and regulate ABA responses in seed germination, root growth and gene expression (Fujii, et al. 2007). Recently, snrk2.2/2.3/2.6 triple mutants were shown to cause a strong ABA insensitive phenotype in seed germination, root growth and gene expression, suggesting that SnRK2.2, SnRK2.3 and OST1/SnRK2.6 have overlapping functions in ABA signaling (Fujii and Zhu 2009; Nakashima et al. 2009).

The dominant ABA insensitive mutants abi1-1 and abi2-1 exhibit ABA insensitivity in seed germination and root growth responses (Koornneef et al. 1984). ABI1 and ABI2 encode type 2C protein phosphatases (PP2Cs) (Leung et al. 1994; Meyer et al. 1994; Leung et al. 1997; Rodriguez et al. 1998). The Arabidopsis genome includes more than 76 PP2C genes (Schweighofer et al. 2004; Xue et al. 2008). Signal transduction analysis in guard cells showed that abi1-1 impairs ABA signaling mechanisms including ABA-activation of S-type anion channels (Pei et al. 1997), ABA-induced [Ca2+]cyt elevations (Allen et al. 1999), ABA-induced Ca2+ channel activation and ROS production (Murata et al. 2001) and ABA activation of the OST1 protein kinase in Arabidopsis (Mustilli, et al. 2002). These findings led to the model that ABI1 PP2C functions very early in ABA signal transduction (Pei, et al. 1997; Allen, et al. 1999; Murata, et al. 2001; Mustilli, et al. 2002). These signal transduction studies prompted us to search for in vivo ABI1-interacting proteins via protein complex purifications in the present study to identify possible redundant early ABA signal transduction proteins.

Studies have shown that six of the nine Arabidopsis PP2Cs belonging to cluster A of the PP2Cs family (Schweighofer, et al. 2004), ABI1, ABI2, HAB1, HAB2, AHG1 and PP2CA/AHG3 function as negative regulators of ABA signaling and have independent and overlapping functions (Gosti et al. 1999; Merlot et al. 2001; Leonhardt et al. 2004; Saez et al. 2004; Kuhn et al. 2006; Saez et al. 2006; Yoshida et al. 2006b; Nishimura et al. 2007; Rubio et al. 2009). Yeast two hybrid screens have identified substrates of ABI1, including the homeodomain transcriptional factor ATHB6 (Himmelbach et al. 2002), the protein kinases, CIPK15, CIPK20 and OST1 (Guo et al. 2002; Ohta et al. 2003; Yoshida et al. 2006a), gluthathione peroxidase3 (Miao et al. 2006) and a homolog of the yeast SWI/SNF chromatin-remodeling complex, SWI3B (Saez et al. 2008).

Recently two independent groups have reported that two Bet VI family proteins, PYR1 and RCAR1, interact with ABI1, ABI2 and HAB1 (Ma et al. 2009; Park et al. 2009). These studies have provided evidence that the recombinant protein PYR1 and the RCAR1-ABI2 protein complex, may function as ABA receptors in ABA signaling (Ma, et al. 2009; Park, et al. 2009). Two dimensional NMR analyses of PYR1 showed that ABA causes shifts in the conformation of PYR1 (Park, et al. 2009). Furthermore, ABA stimulates the interaction of PYR1 with PP2Cs and this ABA-dependent interaction down regulates PP2C activity in vitro (Park, et al. 2009). RCAR1 also negatively regulates PP2C activity after ABA treatment in vitro, though RCAR1 constitutively interacts with PP2Cs. However, whether PYR/PYL/RCAR proteins interact with ABI1 in Arabidopsis remains unknown.

Here, to identify unknown redundant early ABA signaling proteins in vivo, we pursued YFP-ABI1 complex affinity column-based purifications from Arabidopsis plants. Interestingly, we identified nine of the 14 PYR/PYL/RCAR family members as the most robustly co-purified proteins with ABI1. These data show ABI1 interaction with multiple members of PYR/PYL/RCAR family in Arabidopsis.

Here we further address the following emerging questions: (1) Does the ABI1 protein phosphatase interact with the PYR1 protein in vivo and does ABA affect this interaction in vivo and within which time frame? (2) Does ABI1 interact with SnRK2.2, SnRK2.3 and OST1/SnRK2.6 in plants? (3) Does PYR form complexes with these ABA signaling SnRK2 kinases and does ABA affect this interaction? and (4) Do PYR/PYL/RCAR function in ABA-induced stomatal closure and ABA-inhibition of stomatal opening?

Results

Isolation of YFP-ABI1 over-expression plants

To assess further the ABA signaling cascade, we pursued experiments to identify ABI1-interacting proteins in Arabidopsis using affinity column-based protein complex purifications. We generated transgenic YFP-ABI1 and YFP Arabidopsis expression lines in an abi1-3 knockout mutant background (Saez, et al. 2006). Five independent transgenic YFP-ABI1 expression lines were isolated. Western blot and fluorescence microscopy analyses showed that these Arabidopsis lines express the YFP-ABI1 and YFP proteins, respectively (Figures 1a, b). Interestingly, YFP-ABI1 expression plants showed growth and ABA insensitive phenotypes that were comparable to the dominant abi1-1 mutant. 5-week old YFP-ABI1 plants were significantly smaller in size than control YFP expression plants (Figure 1b). Previous research has reported that ABI1-GFP over-expressing lines do not show any ABA response phenotypes compared to vector control lines (Moes et al. 2008). In our investigations, YFP-ABI1 fusion expression plants showed ABA insensitive phenotypes during seed germination, root growth and in stomatal responses compared to the control YFP expression plants (Figures 1c-e).

Figure 1. Constitutive expression of YFP-ABI1 causes ABA insensitivity.

Figure 1

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(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: nonspecific 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 d and then transferred to MS plates containing the indicated ABA concentrations. Root length was measured 4 d 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.

Identification of ABI1-interacting proteins

Using YFP-ABI1 and YFP expression in the abi1-3 knock-out background, we purified ABI1-interacting proteins. A GFP affinity column was loaded with whole protein extracts from YFP-ABI1 and control YFP expression plants grown on MS plates for 21 days with or without ABA treatment. Affinity purified protein complexes were identified by mass spectrometric analyses. The specificity of the proteins purified by YFP affinity purification was analyzed in parallel negative control experiments using YFP expression plants in the abi1-3 mutant background (Tables S1, S2). Upon silver staining, some visible bands overlapped with controls, and specific bands associated that YFP-ABI1 samples were also consistently observed (Figure S1). Mass-spectrometrical analyses of five samples without ABA treatment (four independent samples and one duplicate) and five samples treated with ABA (three independent samples and two duplicates) allowed identification of proteins that associated with YFP-ABI1. Interestingly, the identified proteins included known ABA signaling components SnRK2.2, SnRK2.3, RPN10 and OST2/AHA1 (Tables 1, 2, Tables S5, S6) (Smalle et al. 2003; Fujii, et al. 2007; Merlot et al. 2007). SnRK2.2 and SnRK2.3 were not identified in any of the YFP control experiments (Table S2).

Table 1.

Known ABA signaling proteins co-purified with ABI1 from Arabidopsis plants without addition of exogenous ABA.

Sequence coverage (%)
Symbol Locus AGI Ex1 Ex2 Ex3 Ex4 Ex5 Average
ABI1 AT4G26080.1 AT4G26080 90.6 87.1 83.2 87.1 87.6 87.1 ± 2.6
SnRK2.2 AT3G50500.1 AT3G50500 22.4 13.5 18.0 ± 6.3
SnRK2.3 AT5G66880.1 AT5G66880 8.9 12.5 16.6 12.7 ± 3.9
RPN10 AT4G38630.1 AT4G38630 10.6 14.0 7.3 11.7 10.9 ± 2.8
OST2 AT2G18960.1 AT2G18960 5.6 12.9 2.8 7.1 ± 5.2
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Table 2.

Known ABA signaling proteins co-purified with ABI1 from Arabidopsis plants exposed to exogenous ABA.

Sequence coverage (%)
Symbol Locus AGI Ex6 Ex7 Ex8 Ex9 Ex10 Average
ABI1 AT4G26080.1 AT4G26080 63.4 85.5 89.4 84.6 82.3 81.0 ± 10.2
SnRK2.2 AT3G50500.1 AT3G50500 13.5 16.6 26.0 18.7 ± 6.5
SnRK2.3 AT5G66880.1 AT5G66880 13.6 11.4 19.9 15.0 ± 4.4
RPN10 AT4G38630.1 AT4G38630 14.5 11.7 13.7 7.3 11.8 ± 3.2
OST2 AT2G18960.1 AT2G18960 3.9 10.3 7.1 ± 4.5
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Analyses of mass-spectra identified nine proteins in the absence of exogenously applied ABA that were identified in all YFP-ABI1 experiments, but not in YFP controls (Table 3, Tables S3, S7). In the presence of exogenous ABA, twenty proteins were identified in all YFP-ABI1 samples (Table 4, Tables S4, S8). Initially, we named these proteins candidate ABI1 interacting proteins (ABIP). The protein with the largest sequence coverage was ABI1 (Table 3). Six of the nine proteins in samples that were not treated with exogenous ABA encoded proteins from a, at the time uncharacterized, Bet VI subfamily of proteins (Table 3, Table S3). These Bet VI family proteins including PYR1 and other close homologs have recently been independently identified as candidate ABA-binding and ABA signaling proteins (Ma, et al. 2009; Park, et al. 2009). Interestingly the proteins co-purified with ABI1 with the highest total and unique sequence coverage and number of unique peptides and spectrum count were all members of the PYR/PYL/RCAR protein family (Figure 2a, Table 3, Tables S3, S7). Two additional proteins from the 14 member PYR/PYL/RCAR protein family were identified in three or four of the five experiments (Table 3, PYL7 & PYL10). Moreover, in ABA-treated samples eight PYR1/PYL/RCAR proteins had the largest total and unique sequence coverage and number of unique peptides and spectrum count of the isolated ABI1-interacting proteins and were identified in all five LC-MS/MS experiments (Table 4, Tables S4, S8). As Bet VI proteins were the most significantly and consistently represented proteins from among all sequenced protein peptides in YFP-ABI1 samples, but not in YFP control samples (Table S2), these findings provide evidence that the ABI1 protein phosphatase can form complexes with several of the 14 PYR/PYL/RCAR Bet VI family members in vivo (Tables 3, 4, Figure 2a, Tables S1, S3, S4, S7, S8).

Table 3.

Candidate ABI1-interacting proteins with the largest mass-spectrometrical sequence coverage from five LC-MS/MS experiments analyzing ABI1 complexes isolated from Arabidopsis in the absence of exogenous ABA.

Sequence coverage (%)
Locus AGI Symbol Ex1 Ex2 Ex3 Ex4 Ex5 Average
ABI1 AT4G26080.1 AT4G26080 ABI1 90.6 87.1 83.2 87.1 87.6 87.1 ± 2.6
ABIP1 AT5G53160.2 AT5G53160 PYL8 78.2 63.3 79.8 61.2 64.9 69.5 ± 8.8
ABIP2 AT2G38310.1 AT2G38310 PYL4 87.9 72.5 55.1 40.6 62.8 63.8 ± 17.8
ABIP3 AT5G05440.1 AT5G05440 PYL5 74.4 63.5 71.9 35.5 57.1 60.5 ± 15.7
ABIP4 AT1G01360.1 AT1G01360 PYL9 67.9 50.3 44.9 62.6 59.9 57.1 ± 9.4
ABIP5 AT2G40330.1 AT2G40330 PYL6 57.7 22.3 48.4 28.4 34.0 38.2 ± 14.6
ABIP6 AT4G17870.1 AT4G17870 PYR1 39.8 19.4 36.1 28.8 48.2 34.5 ± 10.9
ABIP11 AT3G53430.1 AT3G53430 RPL12B 28.3 15.1 31.3 5.4 5.4 17.1 ± 12.3
ABIP12 AT5G20280.1 AT5G20280 ATSPS1F 9.2 4.9 9.4 5.0 4.0 6.5 ± 2.6
ABIP7* AT4G01026.1 AT4G01026 PYL7 46.9 37.0 25.1 26.1 33.8 ± 10.3
ABIP8* AT4G27920.1 AT4G27920 PYL10 15.3 7.7 7.7 10.2 ± 4.4
ABIP9* AT5G46790.1 AT5G46790 PYL1 22.6 22.6 ± 0.0
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*

Three additional PYR/PYL/RCAR family members identified in 4, 3 and 1 of the experiments, respectively.

Table 4.

Candidate ABI1-interacting proteins with the largest mass-spectrometrical sequence coverage from five LC-MS/MS experiments analyzing Arabidopsis ABI1 complexes exposed to exogenous ABA.

Sequence coverage (%)
Locus AGI Symbol Ex6 Ex7 Ex8 Ex9 Ex10 Average
ABI1 AT4G26080.1 AT4G26080 ABI1 63.4 85.5 89.4 84.6 82.3 81.0 ± 10.2
ABIP2 AT2G38310.1 AT2G38310 PYL4 50.7 67.6 83.1 83.1 61.8 69.3 ± 14.0
ABIP6 AT4G17870.1 AT4G17870 PYR1 71.2 73.3 70.7 62.8 42.9 61.2 ± 12.5
ABIP1 AT5G53160.2 AT5G53160 PYL8 62.2 71.8 69.7 50.0 42.0 59.1 ± 12.8
ABIP9 AT5G46790.1 AT5G46790 PYL1 55.2 70.6 73.3 41.2 18.1 51.7 ± 22.8
ABIP4 AT1G01360.1 AT1G01360 PYL9 27.3 77.0 61.5 45.5 32.6 48.8 ± 20.6
ABIP3 AT5G05440.1 AT5G05440 PYL5 30.0 43.3 83.3 42.4 34.5 46.7 ± 21.2
ABIP7 AT4G01026.1 AT4G01026 PYL7 12.3 64.9 69.7 49.3 17.5 42.7 ± 26.6
ABIP5 AT2G40330.1 AT2G40330 PYL6 32.1 42.3 52.1 30.7 23.3 36.1 ± 11.2
ABIP13 AT3G19390.1 AT3G19390 MLD14.3 13.7 21.9 7.3 13.3 13.3 13.9 ± 5.2
ABIP14 AT2G20580.1 AT2G20580 AtRPN1a 2.8 10.7 10.1 5.6 5.6 7.0 ± 3.3
ABIP8* AT4G27920.1 AT4G27920 PYL10 7.7 7.7 ± 0.0
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*

PYL10 was identified in one experiment as shown.

Figure 2. ABA causes ABI1-PYR1 interaction in Arabidopsis.

Figure 2

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(a) Example of raw tandem mass spectrometery 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.

ABI1 interacts with PYR1 in Arabidopsis

It was recently shown that ABA enhances the interaction of YFP-ABI1 with HA-PYR1 transiently expressed in Nicotiana benthamiana leaves (Park, et al. 2009). To assess whether these interactions occur in vivo in Arabidopsis, we performed co-immunoprecipitation experiments in stably transformed YFP-ABI1 and YFP control expression lines using a PYR1 antibody. YFP-ABI1 interacted with PYR1 in Arabidopsis with a strong enhancement upon application of 100 μM exogenous ABA for 48 h (Figure S2). Subsequently, co-immunoprecipitation experiments were performed three hours after exogenous application of 0, 1, 10, and 100 μM ABA. ABA caused a clear enhancement in ABI1-PYR1 interaction in vivo (Figure 2b). To determine the timing of this ABA response in Arabidopsis, co-immunoprecipitation experiments were performed 0, 5, 10, 30 or 60 min after application of exogenous ABA. These experiments showed a clear ABA induction of a YFP-ABI1 and PYR1 interaction within 5 min of ABA treatment in vivo (Figure 2c).

PP2Cs, PYR1 and SnRK2s interact with each other in ABA signaling

ABI1 has been reported to interact with OST1/SnRK2E/SnRK2.6 in yeast (Yoshida, et al. 2006a). However it remains unknown whether this interaction occurs in plant cells and whether ABI1 interacts with other SnRK2s. It also remains unknown whether any ABI1-SnRK2 interactions are regulated by ABA similar to the ABI1-PYR1 interaction. As SnRK2.2 and SnRK2.3 were co-purified with ABI1 in our affinity column purification experiments (Tables 1, 2), we co-expressed YFP-ABI1 with either HA-SnRK2.2, HA-SnRK2.3 or HA-OST1/SnRK2.6 in Nicotiana benthamiana and performed co-immunoprecipitation experiments. YFP-ABI1 constitutively interacted with HA-SnRK2.2, HA-SnRK2.3 and HA-OST1/SnRK2.6 both in the absence and presence of exogenously applied ABA (Figure 3).

Figure 3. ABI1 PP2C co-immunoprecipitates with all three ABA signaling SnRK2 kinases in plants.

Figure 3

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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 d 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.

We analyzed whether PYR1 might co-purify with SnRK2s. We co-expressed YFP-PYR1 with either HA-SnRK2.3 or HA-OST1/SnRK2.6 in Nicotiana benthamiana and performed co-immunoprecipitation experiments. Interestingly, YFP-PYR1 co-immunoprecipitated with HA-SnRK2.3 both in the presence and absence of ABA, but not detectably or very weakly with HA-OST1/SnRK2.6 (Figure 4).

Figure 4. Interaction of PYR1 with SnRK2.3 and OST1.

Figure 4

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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 d 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.

pyr1/pyl1/pyl2/pyl4 quadruple mutant plants exhibit ABA insensitive phenotype in guard cells

The abi1-1 protein impairs ABA signaling upstream of known early ABA responses in Arabidopsis guard cells (Pei, et al. 1997; Allen, et al. 1999; Murata, et al. 2001; Mustilli, et al. 2002). A pyr1/pyl1/pyl2/pyl4 quadruple mutant was recently shown to cause strong ABA insensitivities in seed germination, root growth and ABA-induced gene expression (Park, et al. 2009). Whether PYR/PYL/RCAR genes mediate ABA response in guard cells remains unknown. Public microarray data show that the PYR1, PYL1, PYL2, PYL4, PYL5, PYL7 and PYL8 mRNAs are highly expressed in guard cells and seedlings (Leonhardt, et al. 2004; Goda et al. 2008; Yang et al. 2008) (Figure S4). Stomatal response analyses were performed with pyr1/pyl1/pyl2/pyl4 quadruple mutant plants. pyr1/pyl1/pyl2/pyl4 quadruple mutants showed a strong ABA insensitive phenotype in double-blind ABA-induced stomatal closing and ABA-inhibition of stomatal opening analyses (Figures 5a, b, S5). Studies have provided evidence that Ca2+ signaling functions downstream of the abi1-1 and abi2-1 PP2Cs in guard cells (Allen, et al. 1999; Murata, et al. 2001). Experiments were pursued to determine whether pyr1/pyl1/pyl2/pyl4 quadruple mutant plants impair Ca2+-induced stomatal closing. Repetitive addition and removal of Ca2+ to the extracellular solution bathing leaf epidermes while shifting the K+ equilibrium potential in guard cells, results in Ca2+-induced stomatal closure (Allen et al. 2001). Such imposed extracellular Ca2+ pulses caused similar Ca2+-reactive and Ca2+-programmed stomatal closure responses in both pyr1/pyl1/pyl2/pyl4 quadruple mutant and wild type epidermes (Figure 5c). These results suggest that these PYR/PYL/RCAR proteins are critical for ABA signaling in guard cells upstream of Ca2+ signaling.

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.

Figure 5

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(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 treatement). (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.

Discussion

Studies have suggested that ABI1 functions upstream of all analyzed events in early ABA signal transduction in guard cells (Pei, et al. 1997; Allen, et al. 1999; Murata, et al. 2001; Mustilli, et al. 2002). We hypothesized that redundant proteins may act upstream of ABI1, because forward genetic screens had not identified other upstream loci. Our early attempts to co-purify ABI1-complexed proteins from Arabidopsis, showed that additional purification steps led to loss of many ABI1-associated proteins (I. Mori & J. I. Schroeder unpublished data). A rapid purification method using GFP affinity column-affinity purification, as reported here, was more fruitful for isolating redundant ABA signaling proteins in vivo (Tables 1, 2, 3, 4, Tables S1, S2, S3, S4, S7, S8). Here we identify nine of the 14 PYR/PYL/RCAR gene family proteins as in vivo ABI1-co-purified proteins with the largest sequence coverage and number of unique peptides and spectrum count of all identified proteins (Tables 3, 4, Tables S3, S4, S7, S8, Figures 2, 3). After identifying these Bet VI family proteins as the main co-purified proteins in ABI1 complexes from Arabidopsis, we learned that PYR1 had been independently isolated in a chemical genetics screen as a candidate ABA signaling protein (Park, et al. 2009). In the absence of ABA, PYR1 and PYL4 proteins were co-purified as ABI1-interacting proteins (Table 3, Tables S3, S7), although ABA enhanced the interaction of ABI1-PYR1 (Figure 2) and HAB1-PYL4 (Park, et al. 2009). Co-purification results in the absence of exogenously applied ABA may be due to the known endogenous ABA levels in Arabidopsis plants (Nambara and Marion-Poll 2005). On the other hand, ABI1 seems to interact constitutively with PYL5 to PYL9 in Arabidopsis, in the absence of ABA, based on yeast two hybrid data (Ma, et al. 2009; Park, et al. 2009). Over-expression of several other PP2Cs causes ABA insensitive phenotypes in seed germination stages (Saez et al. 2004; Kuhn et al. 2006; Yoshida et al. 2006b; Nishimura et al. 2007). YFP-ABI1 expression plants showed ABA insensitive phenotypes during seed germination, root growth and in stomatal responses compared to the control YFP expression plants (Figures 1c-e).

In addition to the identification of PYR/PYL/RCAR proteins as the major proteins co-purified with ABI1 from Arabidopsis plants, the present study reports several further relevant new findings: (1) A new PYR1 antibody was used and showed that the ABI1 protein phosphatase interacts with the PYR1 protein in vivo and ABA stimulates this interaction in Arabidopsis within 5 min of ABA exposure. (2) ABI1 co-immunoprecipitates with the SnRK2.2, SnRK2.3 and OST1/SnRK2.6 protein kinases in plants, extending previous yeast two hybrid findings (Yoshida, et al. 2006a). (3) PYR1 forms complexes with these ABA signaling SnRK2 kinases in plants both in the presence and absence of ABA. (4) PYR/PYL/RCAR genes are critical for ABA-induced stomatal closure and ABA-inhibition of stomatal opening.

Protein phosphorylation and dephosphorylation events in ABA signaling

The protein kinase OST1/SnRK2.6 is phosphorylated within 5 min of ABA application (Mustilli, et al. 2002; Yoshida, et al. 2006a). ABA binds to PYR1 and RCAR1-ABI2 thus down-regulating PP2C activities (Ma, et al. 2009; Park, et al. 2009). In addition, PYR/PYL/RCAR genes are required for ABA-activation of the SnRK2 kinases (Park, et al. 2009). Here ABA is shown to cause PYR1-ABI1 interaction in Arabidopsis, whereas PYR1 forms a constitutive complex with the SnRK2.3 kinase both in the absence and presence of ABA (Figure 4). Whether PYR1 directly binds to SnRK2s remains to be determined. Furthermore, ABI1 constitutively interacts with the SnRK2.2, SnRK2.3 and OST1/SnRK2.6 kinases in planta (Figure 3). These co-immunoprecipitation results support the hypothesis that these major ABA signaling components, PYR/PYL/RCARs, PP2Cs and SnRK2s, may form an ABA signalosome complex, rather than forming a simple sequential PYR1➔ABI1➔SnRK2 signaling pathway. An initial sequential model (Park, et al. 2009) would predict that SnRK2 kinases may only enter into a complex with PYR1 after ABA exposure.

One of the substrates of SnRK2s is a b-ZIP type transcription factor ABRE/ABF (Furihata et al. 2006; Fujii, et al. 2007). Arabidopsis ost2 and slac1 mutants show strong ABA insensitive phenotypes in guard cells and act downstream of the OST1 kinase (Li, et al. 2000; Merlot, et al. 2007; Negi et al. 2008; Vahisalu et al. 2008). We isolated OST2/AHA1 as a candidate ABI1-interacting protein (Tables 1, 2), which would be consistent with the model that the ABA signaling PP2Cs regulate multiple downstream signaling proteins (Pei, et al. 1997; Allen, et al. 1999; Murata, et al. 2001; Furihata, et al. 2006; Kuhn, et al. 2006; Nishimura, et al. 2007; Saez et al. 2008).

PYR/PYL/RCAR family members regulate ABA signaling in guard cells

Several ABA receptors have been reported to impair ABA-induced stomatal closure; CHLH was reported as an ABA receptor but only one stable and available genetic allele cch (Mochizuki et al. 2001) showed a strong ABA insensitive phenotype (Shen et al. 2006). Recently, double mutants in the candidate ABA receptors, GTG1 and GTG2, were shown to cause ABA insensitivity in seed germination, root growth, stomatal movements and ABA-induced gene expression (Pandey et al. 2009). For ABA-induced stomatal closure, gtg1gtg2 double mutants exhibited a limited insensitivity to ABA compared to WT, suggesting that additional ABA receptors are needed to perceive ABA and transduce the ABA signal independently. More recently, the PYR/PYL/RCAR family proteins were identified as new ABA binding and signaling proteins (Ma, et al. 2009; Park, et al. 2009). Here we show that pyr1/pyl1/pyl2/pyl4 quadruple mutant plants exhibit a strong ABA insensitive phenotype in ABA-induced stomatal closing and ABA-inhibition of stomatal opening but not in Ca2+-induced stomatal closing (Figure 5), suggesting that PYR1/PYL1/PYL2/PYL4 function upstream of Ca2+ in ABA signaling and likely also upstream of Ca2+-independent signaling (Allan, et al. 1994; Siegel, et al. 2009). Furthermore PYR1/PYL1/PYL2/PYL4 mediate ABA signaling not only in seed germination and seedling stages (Park, et al. 2009) but also in guard cells (Figure 5).

In conclusion, in vivo affinity column purifications of ABI1 complex proteins provide evidence that the PYR/PYL/RCAR family of proteins are the most abundant and robust in vivo ABI1-complexed interactors in Arabidopsis. ABA rapidly stimulates PYR1-ABI1 interaction in Arabidopsis and additional findings indicate the possible assembly of an early ABA signalosome.

Experimental procedures

Plant materials and growth conditions

The Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col) was used in this study. Plant growth conditions, seed germination and root elongation assays were performed as described previously (Nishimura et al. 2004; Mori, et al. 2006). ABA-induced and repetitive Ca2+ pulse-stomatal movement responses and apertures were analyzed as described previously (Vahisalu, et al. 2008). For ABA-inhibition of stomatal opening, to pre-close stomata, intact leaf epidermes attached to a cover slip were pre-incubated in buffer (10 mM KCl, 7.5 mM iminodiacetic acid, and 10 mM MES/Tris, pH 6.2) for 3 h in the dark and exposed to the indicated ABA concentrations in the light (100 μmol m−2s−1).

Cloning and Construction of transgenic YFP-ABI1 Arabidopsis lines

The full length ABI1 cDNA was cloned into pDONR vector (Invitrogen) and sequenced and then transferred to the destination vectors pH35YG and pEarleyGate 201 by Gateway LR recombination reaction (Invitrogen). Agrobacterium tumefaciens strain GV3101 was used to transform the abi1-3 T-DNA insertion disruption line and wild-type Col-0 with plants by the floral dip method (Clough and Bent 1998). Transgenic plant lines were screened for hygromycin resistance and homozygous T3 lines were isolated.

Affinity column purification of YFP-ABI1 complexed proteins

Identification of proteins by mass spectrometry is described in the Supporting Information methods.

Co-immunoprecipitation in Arabidopsis and Nicotiana benthamiana

For protein extractions, 3-week-old seedlings (1 g) grown on horizontal MS plates were incubated for 2 hours in water before ABA treatment. Co-immunoprecipitations were performed using Dynabeads Protein G (Invitrogen) captured with anti-GFP antibody (abcam) according to the manufacture's specifications. Co-immunoprecipitations in Nicotiana benthamiana were performed as described previously without modifications (Park, et al. 2009).

Supplementary Material

Supp Fig Legends
Supp Fig s1 - s5
Supp Info Methods
Supp Table s1
Supp Table s2
Supp Table s3 - s8

Acknowledgements

We thank Drs. M. Böhmer and D.G. Mendoza-Cozatl for technical advice, Dr. T.H. Kim for critical comments and reading of the manuscript and Drs. I. Mori and M. Ghassemian (UCSD) for pursuing early abi1-1 and ABI1 complex purification attempts in the Schroeder laboratory. We are also grateful to the Arabidopsis Biological Resource Center (ABRC) and Dr. T. Demura (NAIST) for the pH35YG plasmid. Requests for the pyr1pyl1pyl2pyl4 quadruple mutant seeds should be sent to Sean.cutler@ucr.edu. J.C. is an investigator of the Howard Hughes Medical Institute. This research was supported by NIH (GM060396-ES010337) and NSF (MCB0417118) grants to J.I.S., NSF (IOS-0820508) to S.R.C., NIH (R01 GM052413) to J.C. and NIH (P41 RR011823) to J.R.Y.

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Associated Data

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Supplementary Materials

Supp Fig Legends
NIHMS152710-supplement-Supp_Fig_Legends.doc (24KB, doc)
Supp Fig s1 - s5
NIHMS152710-supplement-Supp_Fig_s1_-_s5.pdf (849.7KB, pdf)
Supp Info Methods
NIHMS152710-supplement-Supp_Info_Methods.doc (87KB, doc)
Supp Table s1
NIHMS152710-supplement-Supp_Table_s1.xls (16.3MB, xls)
Supp Table s2
NIHMS152710-supplement-Supp_Table_s2.xls (8.6MB, xls)
Supp Table s3 - s8
NIHMS152710-supplement-Supp_Table_s3_-_s8.doc (310KB, doc)

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