Genes & Genomics (2012) 34: 345-353 DOI 10.1007/s13258-012-0081-1

REVIEW

ABA signal transduction from ABA receptors to ion channels Chae Woo Lim ․ Woonhee Baek ․ Sohee Lim ․ Sung Chul Lee 1)

Received: 05 May 2012 / Accepted: 30 May 2012 / Published online: 13 August 2012 © The Genetics Society of Korea and Springer 2012

Abstract The plant hormone abscisic acid (ABA) is involved in regulating a number of major processes such as seed dormancy, seedling development, and biotic and abiotic stress responses. The function and effect of ABA on pathogens are still unclear, but the roles of ABA in seed germination and abiotic stress responses have been well characterized.Abiotic stresses elevate ABA levels and activate ABA signaling; thus, inducing a variety of responses, including the expression of stress-related genes and stomatal closure. The past decade has witnessed many significant advances in our understanding of ABA signal transduction due to application of a combination of approaches including genetics, biochemistry, electrophysiology, and chemical genetics. A number of proteins associated with the ABA signal transduction pathway such as PYR/PYL/RCAR family of START proteins, have been identified. These ABA receptors bind to ABA and positively regulate ABA signaling via inactivation of PP2C phosphatase activity, which inhibits SnRK2-type kinases by direct interaction and dephosphorylation. Additionally, SnRK2-type kinases and PP2Cs interact with one another and with other components of ABA signaling and function as positive and negative ABA regulators, respectively. In this review, we focus on ABA function to abiotic stresses and highlight each component in relation to ABA and its interactions. Keywords ABA receptor; Abiotic stress; SnRK2-type kinase; 2C-type protein phosphatase; Ion channel

Introduction Plants activate a complex series of defense mechanism that lead to survivalin response to biotic and abiotic stresses.The plant hormone abscisic acid (ABA) functions in a variety of C. W. Lim ․ W. Baek ․ S. Lim ․ S. C. Lee ( ) School of Biological Sciences (BK21 program), Chung-Ang University, Seoul 156-756, Korea e-mail: [email protected]

ways during plant development such as inhibition of seed germination and lateral root elongation, and enhanced tolerance to environmental stresses and pathogen attack(Ton et al., 2009; Robert-Seilaniantz et al., 2007; Finkelstein et al., 2002). Changes in ABA concentration and sensitivity, which are triggered by biotic and abiotic stresses, mediate adaptive plant responses. Drought, high salt, and cold are common environmental stresses that adversely affect plant growth and induce severe losses in crop production. Plants lose their water primarily through leaf stomata. The physiological and molecular mechanisms involved in such water stresses have been widely studied (Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006). However, the osmotic stress responses of plants are complex phenomena, and the exact structural and functional modifications induced by these osmotic stresses are currently poorly understood. Osmotic stress can be a common consequence of exposure to high salt, drought,and low temperature stresses (Hasegawa et al., 2000; Apse and Blumwald, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006), and protection against osmotic stress causedby drought and high salinity depends on minimizing stomatal waterloss from leaves and maximizing water uptake by roots. ABA plays a crucial role in reducing water loss by regulating the stomata opening. The function of ABA in these osmotic stress responses has been extensively studied and reviewed (Wilkinson and Davies, 2010; Assmann, 2003; Hubbard et al., 2010; Popko et al., 2010; Cutler et al., 2010; Wasilewska et al., 2008). ABA is synthesized in response to osmotic stress conditions, which promote ABA biosynthesis and the distribution of ABA in the plant body, including guard cells (Wilkinson and Davies, 2002). Additionally, several physiological changes in response to various abiotic stresses result from ABA-induced changes in stress-related gene expression patterns, which ultimately lead to variousadaptive responses at the cell and whole plant levels(Ramanjulu and Bartels, 2002; Shinozaki and Yamaguchi-Shinozaki, 2000). Stomatal closure has several effects including water conservation, inhibition of photosynthesis, and exclusionof pathogens. If plants lose more water via the stomata than is

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taken up by the root, their tissues can be damaged, resulting in cell death. The primary function of stomatal closure is to prevent water loss, which induces drought tolerance in plants. ABA-controlled processes are necessary for plant survival, and ABA-deficientmutants are susceptible to water stress(Finkelstein et al., 2005; Kang et al., 2002). In contrast, the roles of ABA in plant defense responses against pathogen infection are still poorly understood and even controversial (Anderson et al., 2004; Ton and Mauch-Mani, 2004). However, recent studies have shown that ABA functions as an essential signal in plant immunity to pathogens(Adie et al., 2007; Melotto et al., 2008; Melotto et al., 2006). In this review, we summarize the role of ABA by integrating plant responses to abiotic stresses.

ABA receptors in ABA signaling Both positive and negative regulators of ABA signaling, including protein kinases, phosphatases, and ion channels have been identified (Assmann, 2003; Fujii et al., 2007; Fujii and Zhu, 2009; Geiger et al., 2009; Gosti et al., 1999; Hosy et al., 2003; Kwak et al., 2002; Lee et al., 2009; Merlot et al., 2001). However, research on ABA receptors is currently insufficient. Using biochemical approaches, ABA-binding activities have been observed in several cell compartments, such as the cellular membrane and cytosol, suggesting that various ABA receptors exist (Kitahata et al., 2005; Pedron et al., 1998; Zhang et al., 2001a).Several research groups have focused on ABA receptors, although most of their functions remain unconfirmed (Park et al., 2009; Ma et al., 2009; Christmann and Grill, 2009; Pandey et al., 2009; Liu et al., 2007; Shen et al., 2006). Here, we will discuss the G-protein coupled receptors (GPCRs) and the PYR/PYL/RCAR family of ABA receptors, which are relatively well characterized. GPCRs, which include some ABA receptors, have been analyzed using biochemical and bioinformatic methods (Liu et al., 2007; Pandey et al., 2009; Chen and Jones, 2004; Chen et al., 2003). G proteins are composed of Gα, Gβ, and Gγ subunits and function as signal transducers (Offermanns, 2003). GPCRs receive extracellular signals at the cellular membrane (Pierce et al., 2002). GCR1 was the first identified GPCR in Arabidopsis and regulates ABA signaling, whereas its ligand has not been identified (Plakidou-Dymock et al., 1998; Pandey and Assmann, 2004; Colucci et al., 2002; Assmann, 2005). Additionally, Liu et al. (2007) found that the GCR2 protein, which is a GPCR from Arabidopsis, binds to ABA with high affinity and is localized in the plasma membrane. In addition, gcr2 mutants show an ABA insensitive phenotype for germination and stomatal closure (Liu et al., 2007). However, other research groups could not find any evidences that GCR2 protein functions as ABA receptor (Gao et al., 2007; Guo et al., 2008; Risk et al., 2009).

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Recently, GCPR type G proteins (GTG1 and GTG2), which are homologous to an orphan vertebrate GPCR (GPR89), have been isolated in Arabidopsis as membrane-localized ABA receptors through bioinformatics analysis (Pandey et al., 2009). These two proteins specifically bind ABA, but only 1% of the GTG proteins have binding activity (Pandey et al., 2009). GTGs have a nucleotide binding domain, which is unlike other GPCRs such as GCR2 and GCR89. When GTG proteins bind GDP, the receptors have high affinity for ABA (Pandey et al., 2009). The double mutants (gtg1/gtg2) display an insensitive ABA phenotype, including increased germination rate and seedling development in the presence of ABA. However, further studies are required to validate the GTG proteins as ABA receptors. The pyrabactin resistance (PYR)/PYR-like (PYL)/regulatory component of the ABA receptor (RCAR) has been recently isolated as an intracellular ABA receptor family, which includes 14 members (Ma et al.,2009; Park et al., 2009; Santiago et al., 2009b). Genetic analyses have revealed that triple (pyr1:pyl1:pyl4) and quadruple (pyr1:pyl1:pyl2:pyl4) mutants show ABA insensitivity in seed germination, root growth, and stomatal closure (Park et al., 2009; Nishimura et al., 2010). In addition to this ABA insensitivity, ABA-inducing gene expression iscompromised in the mutants compared to that in wild-type plants (Park et al., 2009). Forward and reverse genetic methods have been used to identify the downstream target proteins of PYR/PYLs (Ma et al., 2009; Park et al., 2009). Ma et al. (2009) used ABI2 as bait in a yeast two-hybrid system to detect RCAR1 (PYL9). However, Park et al. (2009) used PYR1, which is necessary for the ABA agonist pyrobactin as bait to find ABI1, ABI2, and HAB1. PYR/PYLs directly inhibit phosphatase activity of PP2Cs in vitro (Ma et al., 2009; Szostkiewicz et al., 2010). It has already been determined that clade A PP2Cs are negative regulators of ABA (Schweighofer et al., 2004; Gosti et al., 1999; Merlot et al., 2001; Wasilewska et al., 2008). Structural analyses have been conducted on PYR/PYL/ RCAR alone and in complex with ABA and PP2Cs (Nishimura et al., 2009; Yin et al., 2009; Santiago et al., 2009a; Miyazono et al., 2009; Melcher et al., 2009). START proteins harbor a “ligand-binding pocket” conserved domain and function as a receptor for signaling molecules (Schrick et al., 2004; McConnell et al., 2001; Iyer et al., 2001; Lytle et al., 2009). Interactions between PYR1, PYL1–PYL4, and HAB1 are promoted by ABA; however, interactions between PYL5, RCAR1 (PYL9), and HAB1 and ABI1, respectively, are independent of ABA in a yeast two-hybrid assay (Santiago et al., 2009b; Ma et al., 2009). PYR/PYR/RCAR directly interacts with the active site of PP2Cs and inhibits phosphatase activity (Melcher et al., 2009; Miyazono et al., 2009; Yin et al., 2009). These results indicate that PYR/PYLs inactivation of PP2Cs is mediated by ABA (Park et al., 2009; Ma et al., 2009; Szostkiewicz et al., 2010; Santiago et al.,

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2009b)Collectively, the genetic, physiological, and structural data indicate that PYR/PYLs/ RCARs are ABA receptors that regulate ABA signaling.

Protein kinases and phosphatases in ABA signaling Reversible phosphorylation is a critical regulatory mechanism for many biological processes in all organisms, including plants. The phosphorylation state of a protein is controlled dynamically by protein kinases and phosphatases. These protein kinases and phosphatases are regulated at the levels of gene expression, cellular localization, substrate specificity, and enzyme activities. The Arabidopsis genome contains 1,085 protein kinases, which are the most abundant proteins, and comprise approximately 4% of encoding genes (Hrabak et al., 2003). Among these kinases, both calcium dependent protein kinase (CDPK) and Snf1-related kinase (SnRK), which are calcium-dependent and -independent protein kinases, respectively, are associated with ABA signaling. The Arabidopsis genome encodes 38 SnRKs, and the SnRK family consists of three subgroups, including SnRK1, SnRK2, and SnRK3 based on sequence similarity and domain structure (Halford and Hey, 2009; Hrabak et al., 2003). SnRK1 members have been associated with yeast and animals, whereas SnRK2 and SnRK3 have been reported only in plants (Hrabak et al., 2003). The SnRK2 type kinase family includes 10 members and is divided into three subclassess in Arabidopsis (Kobayashi et al., 2004). Among them, the kinase activity of SnRK2.2/SnRK2.3/SnRK2.6 (OST1) is activated by ABA (Fujii et al., 2007; Mustilli et al., 2002). Based on phenotypic analyses, the ost1 mutant displays an ABA insensitive phenotype by opening stomata under drought conditions (Yoshida et al., 2002; Mustilli et al., 2002). The triple mutant (snrk2.2/snrk2.3/snrk2.6) displays a severe phenotype to water stress and ABA signaling(Fujii and Zhu, 2009; Fujita et al., 2009; Nakashima et al., 2009). The ost1 mutant is not different from wild-type plants withregard to seed dormancy and germination (Mustilli et al., 2002; Yoshida et al., 2002), whereas the snrk2.2/snrk2.3 double mutant has an ABA insensitive phenotype withregard to seed germination and root growth (Fujii et al., 2007). Additionally, ABF2 and ABF3, which are transcription factors that bind to ABA responsive elements, interact with OST1 leading to ABA regulated gene expression (Fujita et al., 2009; Furihata et al., 2006; Sirichandra et al., 2010; Fujii et al., 2009). Sirichandra et al., (2010) also suggested that phosphorylation of ABF3 by OST1 creates the 14-3-3 protein binding motif and stabilizes ABF3 to sustain ABA-regulated gene expression. ABFs, including ABF3 and ABF4, are also involved in stomatal closure in Arabidopsis. ABF3 and ABF4 overexpression reduces transpiration and enhances the drought tolerant phenotype (Kang et al., 2002).

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However, OST1 is associated with ABA-independent responses in sucrose synthesis and fatty acid desaturation (Zheng et al., 2010), suggesting that OST1 is involved in signaling pathways other than ABA signaling. The rice genome encodes 10 members of the SnRK2 family (SAPK) but only three members, including SAPK8, SAPK9, and SAPK10 are induced by ABA (Kobayashi et al., 2004; Hrabak et al., 2003). SAPK8, SAPK9, and SAPK10 are orthologues of SnRK2.2, SnRK2.3, and SnRK2.6 (Hrabak et al., 2003). These SAPK kinases are also activated by ABA and phosphorylated TRAB1, a rice ABF transcription factor, in an ABA-dependent manner (Kobayashi et al., 2005). These results indicate that the SnRK2-type kinases in both Arabidopsis and rice are positive regulators of ABA signaling and water stress (Kobayashi et al., 2005; Nakashima et al., 2009; Fujita et al., 2009; Fujii and Zhu, 2009; Kobayashi et al., 2004). Protein phosphatases are divided into two major classes: protein serine/threonine phosphatases and protein tyrosine phosphatases. Protein serine/threonine phosphatases are classified into the phosphoprotein phosphatase (PPP) [PP (protein phosphatase) 1, PP2A, PP2B, PP4, PP5, PP6 and PP7] and PPM (metallo-dependent protein phosphatase; PP2C) gene families (Cohen, 1997; Das et al., 1996). The PPP and PPM family members are unrelated in sequence, but the structural folds at the catalytic center are quite similar (Das et al., 1996; Ache et al., 2000). PP2Cs function as negative regulators of the stress-signal transduction pathwayin yeast and mammals. In Arabidopsis, 76 genes have been identified as PP2Cs among 112 phosphatases, and nine genes (clade A) are associated with ABA signaling (Schweighofer et al., 2004; Kerk et al., 2002). Here, we will focus on PP2Cs in ABA signal transduction. Using genetic analysis, clade A PP2Cs, including ABI1 and ABI2, act as negative regulators of ABA(Rubio et al., 2009; Gosti et al., 1999; Merlot et al., 2001; Wasilewska et al., 2008). Additionally, HAB1, HAB2, AHG1, and PP2CA have been identified in seed germination mutants,indicating that they display a hypersensitivity response to ABA and functionas negative regulators of ABA (Robert et al., 2006; Saez et al., 2004; Rubio et al., 2009; Kuhn et al., 2006; Nishimura et al., 2004; Saez et al., 2006; Yoshida et al., 2006b). The AHG1 and PP2CA mutants display a hypersensitivity phenotype to ABA during germination and seedling growth, but the agh1-1 mutant displays no ABA-related phenotype in adult plants (Yoshida et al., 2006b; Nishimura et al., 2007; Kuhn et al., 2006), indicating that AHG1 has roles only during germination and early development. Additionally, PP2Cs are also associated with expression of ABA-related genes. PP2CA overexpression inhibits the transcription of ABA-response genes in maize mesophyll protoplasts, and ABI1 interacts with the homeodomain protein AtHB6, which functions downstream of ABA (Himmelbach et al., 2002; Sheen, 1998). The

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clade A PP2Cs function both dependently and independently. The double mutants of hab1-1/abi1-2, abi1-1R4/Abi2-1R1, hab1-1/pp2ca-1, and abi1-2/ pp2ca-1have a more profound insensitive phenotype to ABA when compared with each single mutant (Merlot et al., 2001; Rubio et al., 2009). This demonstrated that there is functional redundancy among these PP2Cs. The physiological function of several SnRK2-PP2C pairs has been determined (Umezawa et al., 2009; Yoshida et al., 2006a; Lee et al., 2009; Fujii et al., 2009; Vlad et al., 2009). The interaction of PP2CA-OST1, which was demonstrated throughin vivo and in vitro assays, may play a role in stomatal closure via phosphorylation and dephosphorylation of the SLAC1 anion channel (Lee et al., 2009; Geiger et al., 2009). The other interaction between ABI1 and OST1 functions in stomatal closure. Hubbard et al. (2010) suggested that the earliest processes occurring in the ABA signal transduction pathway involve interactions between PYR/PYL/RCARs, PP2Cs, and SnRK2s. The interaction between PP2C and SnRK2 results in PP2C-dependent negative regulation, but the participation of PYR/PYL/RCARs in this interaction results in activation of ABA signal transduction (Ma et al., 2009; Park et al., 2009; Umezawa et al., 2009; Fujii et al., 2009). These results suggest that the ABA signal transduction pathway is highly controlled from the ABA receptor to the negative and positive regulators. The triple mutant abi1-2/hab1-1/pp2ca-1results in constitutive activation of SnR2.2, SnRK2.3, and OST1 (Fujii et al., 2009), indicating that the ABA dependency of SnRK2 type kinase activation can be attributed to PP2Cs. There are 14 members of PYR/PYL/RCAR receptors, 6–9 members of clade A PP2Cs, and three members of the SnRK2s, which interact with one another. Therefore, more than 200 combinations exist, and each combination regulates downstream transduction pathways slightly differently via variable ABA concentrations and locations, although some combinations are associated with the same signaling pathway. PP2Cs may interact with different substrates. AMP-activated kinase (AMPK) is a HAB1 substrate inphosphopeptide assays, and the SWI3B subunit of the SWI/SNF chromatin-remodeling complex interacts with HAB1 (Saez et al., 2008). Additionally, ABI1 and ABI2 interact with other proteins. ABI1 interacts with a number of other ABA signaling components, including the ATHB6 transcription factor (Himmelbach et al., 2002), stress-activated MPK6 kinase (Leung et al., 2006), and CBL-associated protein kinases (CIPK) (Ohta et al., 2003; Guo et al., 2002). Additionally, ABI2 physically interacts with CIPK24 (SOS2), which is a member of the SnRK3 family related to salt tolerance, and the abi2-1 mutant exhibits a salt-tolerant phenotype (Ohta et al., 2003). Thesedata suggest that PP2Cs function as negative regulators of ABA and carry out a variety of functions regulating other signaling components.

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ABA signaling to ion channels and guard cells Guard cells regulate the stomatal aperture to limit transpiration via ABA signaling, which involves several ion channels in the plasma membrane and tonoplasts(Schroeder et al., 2001). The ion channels involved in the stomatal aperture include the S-type anion channels and inward- and outward-rectifying K+ channels (Lemtiri-Chlieh and MacRobbie, 1994; Negi et al., 2008; Vahisalu et al., 2008; Schroeder et al., 1987; Schroeder and Hagiwara, 1989). When ABA level increases, anion efflux occurs within the anion channel inducing depolarization(Schroeder and Keller, 1992; Levchenko et al., 2005). Depolarization drives K+ efflux through outward-rectifying K+ channels and the exportation of anions/cations drives water efflux(Ward et al., 1995; Becker et al., 2003). Additionally, water efflux also reduces guard cell volume and induces stomatal closure(Ward et al., 1995; Wasilewska et al., 2008). The regulation of stomatal closure is associated with several factors, anda change in pH is one of the key factors in the regulation of stomatal closure. ABA increases the pH in guard cells, which activates the outward-rectifying K+ channels, whereas reduced pH inactivates the inward-rectifying K+ channels(Blatt and Thiel, 1994; Miedema and Assmann, 1996). The regulation of stomatal closure has also been associated with reactive oxygen species (ROS) and nitric oxide (NO). ROS and NO are generated in response to ABA in guard cells and are associated with stomatal closure (Neill et al., 2002; Pei et al., 2000; Zhang et al., 2001b; Kwak et al., 2003; Neill et al., 2008; Desikan et al., 2002). The PP2Cs, ABI1 and ABI2, are also associated with ROS generation and play a role in the regulation of stomatal closure (Murata et al., 2001). Using ABA insensitive mutants, the roles of ABI1 and ABI2 were shown to reside downstream of ROS and NO production (Desikan et al., 2002; Murata et al., 2001). Additionally, genetic studies have demonstrated that NO production occurs downstream of ROS (Bright et al., 2006) and that NO acts on the inward-rectifying K+ channels and anion channels in guard cells, which induce stomatal closure (Garcia-Mata et al., 2003). Furthermore, the regulation of anion channels is associated with protein kinases and PP2Cs, including CDPK, OST1, ABI1, ABI2, and PP2CA (Geiger et al., 2010; Geiger et al., 2009; Lee et al., 2009; Pei et al., 1997). Protein kinases such as OST and CDPK phosphorylate and activate anion channels, whereas PP2Cs inactivate anion channels via interactions and dephosphorylationof kinases and channels (Lee et al., 2009; Geiger et al., 2010; Geiger et al., 2009).

Concluding Remarks The plant hormone ABA is associated with the regulation of complex signaling pathways involved with seed dormancy, de-

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Figure 1. Working model of how the abscisic acid (ABA) signaling pathway regulates stomatal closure and gene expression through ABA signal transduction. The ion channels and ABFs are regulated by protein-protein interactions and phosphorylation leading to stomatal closure.

velopment, and responses to biotic/abiotic stresses. The function of ABAin abiotic stress is more organized than that of biotic stress. The ABA signaling pathway has several key components. Each component functions as a positive and negative regulator at each step. The best examples of this are the PYR/PYL/RCAR, PP2Cs, SnRKs, and SLAC1 channel. The PYR/PYL/RCAR ABA receptors bind to ABA and inhibit clade A PP2Cs. PP2Cs inhibit SnRKs via a direct interaction and dephosphorylation, and the SnRKs activate bZIP transcription factors and channels via phosphorylation (Fig. 1). Figure 1 shows a model of the ABA signal transduction pathway in guard cells via the physiological responses. Diverse correlations have been derived regarding the effects of ABA on biotic and abiotic stresses. At one point, the level of ABA increases, and tolerance to abiotic stress also increases, but resistance to pathogensdecreases. However, at other times, the accumulation of ABA boosts defense responses to both biotic and abiotic stresses. The exact function of ABA and the ABA signaling pathway model will be further understood through additional studies. A better understanding of how ABA functionsin response to abiotic stresses is important to design effective strategies for engineering abiotic stress-tolerant crops.

Acknowledgements This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ0082222),Rural Development Administration, and the Research Foundation of Korea (NFR) grant funded by the Korea government (MEST) (NO. 2011-0029568) and the Chung-Ang University Research Grants in 2010.

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