E. Grill, Department of Botany, Technische Universität München, Emil-Ramann-Strasse 4, 85354 Freising, Germany Fax: +49 8161 715432 Tel: +49 8161 715433 E-mail: email@example.com
Type 2C protein phosphatases (PP2Cs) form a structurally unique class of Mg2+-/Mn2+-dependent enzymes. PP2Cs are evolutionary conserved from prokaryotes to higher eukaryotes and play a prominent role in stress signalling. In this review, we focus on the evolution, function and regulation of the plant PP2Cs. Members of a subclass of plant PP2Cs counteract mitogen-activated protein kinase pathways, whereas members of other subfamilies function as co-receptors for the phytohormone abscisic acid. Recent structural analyses of abscisic acid receptors have elucidated the mode of ligand-dependent regulation and substrate targeting.
Embryophytes, also known as land plants, are sessile organisms that have evolved a high degree of developmental and metabolic plasticity to allow the fine tuning of responses to environmental challenges. Land plants have recruited the common toolbox of signalling components such as receptors, protein kinases and phosphatases to sense and transduce a broad range of environmental signals. Plant protein phosphatases are predominantly protein serine/threonine phosphatases that belong to the three classes: phosphoprotein phosphatases, phosphoprotein metallophosphatases and aspartate-based protein phosphatases . The phosphoprotein phosphatases include a number of subfamilies such as heteromeric protein phosphatase 1 and protein phosphatase 2A. The subclasses are distinguished by different dependencies on metal ions and sensitivities to inhibitors such as okadaic acid and microcystin. The phosphoprotein metallophosphatase class is represented by the Mg2+ and Mn2+-dependent type 2C protein phosphatases (PP2Cs). The Arabidopsis genome encodes a small number of different protein phosphatase 1 catalytic and regulatory subunits (14) compared to mammals (> 100) and a comparable number of protein phosphatase 2A catalytic and regulatory subunits (∼ 20–30) [2–5]. Arabidopsis and rice are deficient in PP2B calcineurin-like protein phosphatases , although they possess a large number of PP2Cs compared to mammals [7,8]. PP2Cs are considered to be active as monomers. To our knowledge, no small chemical compound has to date been reported as a specific inhibitor of PP2Cs.
Plant PP2Cs have emerged as major players in stress signalling . The present review updates earlier reviews on plant PP2C evolution and function by emphasizing recent insights into the regulatory mode of PP2C co-receptor function and the role of PP2Cs in stress signalling and plant development [10–12].
Evolution of the protein family
The different subclasses of phosphoprotein phosphatases are structurally related but share no sequence homology with PP2Cs . PP2Cs emerged early in evolution as distinct protein phosphatases and are found in archaea, bacteria, fungi, plants and animals. The function of PP2Cs pertains primarily to stress signalling . In some instances, the functions and modes of PP2C action are unexpectedly conserved throughout the divergent phyla [14–16]. Two PP2Cs of Bacillus subtilis play a central role as regulators of physical and energy stress responses by physically interacting with sensor proteins (Fig. 1A) and mediating the activation of specific regulators of gene expression . In plants, a similar pathway operates to control signalling of the stress hormone abscisic acid (ABA) (Fig. 1A), which is induced in response to drought (see below). PP2Cs of Saccharomyces cerevisiae negatively control the Hog1 pathway (Fig. 1B) by counteracting the mitogen-activated protein kinase (MAPK) pathway of the osmotic stress response . Similarly, PP2Cs of Arabidopsis negatively regulate MAPK pathways involved in stress responses and development (Fig. 1B, see below).
In higher plants, such as Arabidopsis and rice, PP2Cs comprise 80 and 90 members, respectively, belonging to ten or more subgroups (Fig. 2A, A–K) [11,19]. During evolution from prokaryotes to multicellular eukaryotes, PP2Cs have diversified, with an increase in the number of distinct subclades and in the total number of genes for PP2C per genome (Fig. 2B). Subgroup A and B members of plants are found neither in prokaryotes, such as the archaebacterium Thermococcus, the cyanobacterium Synechococcus and the eubacterium B. subtilis, nor in nonplant eukaryotes, such as yeast. Clade A PP2Cs are found in unicellular green algae (e.g. Chlamydomonas reinhardtii), and their number has increased throughout evolution (Fig. 2). Clade B PP2Cs are present in the fern-related plant Selaginalla moellendorffii and in higher plants, whereas they are absent in the green alga Chlamydomonas and the moss Physcomitrella patens. The genomes of C. reinhardtii (0.12 Gbp) , P. patens (0.14 Gbp)  and S. moellendorffii (0.11 Gbp)  are comparable in size to the smaller genomes of higher plants exemplified by Arabidopsis (0.14 Gbp)  and rice (0.46 Gbp) . However, the number of genes for PP2C increased from ten in the green alga Chlamydomonas to ∼ 50 in the moss Physcomitrella and the lycophyte Selaginella, and up to 80–130 different genes for PP2C in Arabidopsis and maize, respectively. The increase and diversification the PP2C genes correlates with the evolution of multicellularity in plants and the development of high plasticity found in land plants allowing integration with ever changing environmental conditions .
Below, we summarize the biological roles and molecular function of plant PP2Cs, focusing primarily on Arabidopsis proteins. Clades A to E are presented separately.
PP2Cs of clade A and abscisic acid signalling
The first plant PP2C was identified as a mutation giving rise to the ABA-insensitive phenotype of the mutant abi1 (ABA insensitive 1) [25,26]. ABI1 and its homologue ABI2 [27,28] control the full range of ABA responses, including the regulation of transpiration, vegetative growth and seed germination . The phytohormone ABA also plays a crucial role in plant responses to abiotic stress factors such as drought, cold and heat. ABA regulates the water status of plants and initiates acclimation responses . It is a sesquiterpenic compound related to retinoic acid, and is derived from carotenoid precursors . Targets of ABA signalling include ion channels and aquaporins, which regulate hydrostatic cell pressure and thereby stomatal aperture [32,33]. Stomatal apertures determine the exchange of CO2, O2 and water vapour between plants and the atmosphere, and thus plant growth and water status. In addition, ABA signalling regulates gene expression by targeting basic-zipper class transcription factors that bind to ABA-responsive cis-elements (Fig. 1A) [34,35].
ABI1 and ABI2 cluster together with seven other PP2Cs in clade A of the Arabidopsis protein family of 80 members [7,36]. The central role of ABI1 and ABI2 in ABA signalling was recognized early on . Subsequently, at least seven members of clade A have emerged as regulators of ABA responses, negatively controlling ABA-invoked physiological responses such as the inhibition of germination and root growth or stomatal closure [37–40]. In addition to prototypical ABI1 and ABI2, clade A contains PP2CA/AHG3 (ABA Hypersensitive Germination 3) , HAB1 (Homology to ABI1 1) , HAB2 (Homology to ABI1 2) , HAI1 (Highly ABA-Induced PP2C 1), HAI2 (Highly ABA-Induced PP2C 2) and HAI3 (Highly ABA-Induced PP2C 3) , as well as AHG1 (ABA Hypersensitive Germination 1) .
The redundant functions of clade A PP2Cs with respect to the regulation of seed germination are reflected by the weak or missing ABA-hypersensitive phenotype generated by the inactivation of single PP2C members . By contrast, multiple clade A PP2C knockouts exhibit a robust ABA hypersensitive phenotype [45,46]. A common feature of clade A PP2Cs is their transcriptional up-regulation in response to high ABA levels or stress conditions that stimulate ABA biosynthesis . It has been postulated that this response desensitizes the plant to high ABA concentrations in a negative-feedback loop . The various PP2C members physically interact with a number of cytosolic and nuclear-localized proteins, emphasizing the multiple roles and targets of the protein phosphatases in the ABA responses [38,49,50]. Several of these interactions, such as binding to the homeodomain transcription factor AtHB6  and CIPK24 , are shared among PP2Cs, including ABI1 and ABI2. Other interactions are more selective, such as the specific interaction between ABI2 and the preprotein of fibrillin, a stress-protective plastidic protein , or with CIPK8 .
Among the interaction partners of clade A PP2Cs, there is a group of proteins structurally related to the pollen allergen Bet v 1 [54–56]. These PP2C binding proteins of ∼ 20 kDa have been identified as soluble ABA-binding proteins and termed ABA co-receptors regulatory component of ABA receptor (RCAR)  and ABA receptor proteins pyrabactin resistance 1 (PYR1)/PYR1-like . In the presence of ABA, RCARs/PYR1-like proteins inhibit protein phosphatase activity.
The Bet v 1 protein superfamily is characterized by its helix-grip fold motif . Small hydrophobic ligands can be bound in the cavity formed by seven β-sheets and one to three α-helices in the typical Bet v 1 class member. Analysis of crystal structures of ABA-free (apo form) and ABA-bound RCAR proteins revealed conformational changes of two loop regions in the presence of ABA [58–61]. The loops β3–β4 and loops β5–β6 of RCARs, also termed ‘gate’ and ‘latch’, engulf the ligand, still leaving an ABA contact site for the protein phosphatase . In addition, some RCARs such as PYR1/RCAR11 are able to bind the sulfonamide pyrabactin, an ABA agonist, at the ABA binding site [56,63]. Site-directed mutational analysis of ligand contacting residues identified amino acid residues critical for discriminating ABA against pyrabactin [64,65]. The phytohormone ABA is contacted also by the PP2C itself in complex with RCAR [66,67]. A specific tryptophan residue, which is conserved among clade A members except for AHG1, physically interacts with ABA via a water molecule . Mutation of this tryptophan residue in HAB1 resulted in an ABA-insensitive PP2C regulation and a drop in the ABA affinity of the receptor complex . In agreement with a PP2C co-receptor function, dissociation constants of ABA from RCARs are one to three orders of magnitude higher than those of the holoreceptor complex [48,55]. Although RCARs bind ABA with affinities ranging from a Kd of ∼ 1–100 μm ABA, half maximal inhibition of different PP2Cs was observed in the range of 10–300 nm ABA [70,71]. The physiologically relevant ABA concentration is in the submicromolar level under nonstress conditions and may reach low micromolar levels under severe stress conditions . The ABA receptor requires the co-receptor ABI1 or other clade A PP2C members to be responsive to submicromolar ABA levels.
Formation of the trimeric complex between the ligand ABA and the holoreceptor abolishes the PP2C-mediated inhibition of ABA signalling. Subfamily 2 of the SNF1-related kinases (SnRK2s) (e.g. OST1) functions downstream of the PP2Cs as a positive regulator of ABA signalling [46,73–76]. Autoactivation and transactivation of these protein kinases results in phosphorylation and activation of downstream targets, ABA-responsive transcription factors belonging to the basic-zipper class (e.g. ABF1 and ABI5) [35,77] and ion channels required for stomatal closure [78–82]. Recent structural insights substantiate the mechanism for basal activity and autoactivation of SnRK2s by conformational changes in their activation loop after release from the PP2C control [83–85]. ABI1 and HAB1 are able to dephosphorylate pSer176 of OST1, which is located in the activation loop and is necessary for autoactivation of the protein kinase [84,85]. Interestingly, binding of RCARs and OST1 in the catalytic cleft of the PP2Cs is coordinated by the amino acid residues that are conserved among clade A members and also conserved in the interacting proteins (Fig. 3) [66,68,86]. The data support the notion that RCARs function as a pseudosubstrate, which becomes locked in the presence of the ABA ligand .
Taken together, biochemical and protein interaction analyses show that RCAR proteins interact with at least eight of the clade A PP2Cs to inhibit the negative regulation of downstream targets [54,88,89] (S. Fuchs, unpublished data). The combinatorial regulation of the respective proteins and the different affinities of the co-receptor moieties for the ABA ligand and the corresponding interaction partners generate a diversified and highly specific machinery for modulating and fine-tuning diverse stress responses throughout the plant life-cycle [90–93].
Although PP2Cs of clade A function as co-receptors of ABA signalling, the unravelling of co-regulatory networks and functional analyses of other PP2Cs suggest that they may also play a role in ABA-dependent stress responses .
Clade B PP2Cs: AP2Cs
Members of clade B have been characterized as regulators of MAPK activities. Clade B contains six genes named AP2Cs (Arabidopsis phosphatase 2C), which are orthologous to Medicago sativa MP2C [95,96]. Four members of this cluster (AP2C1–4) maintain a kinase interaction motif at the N-terminal part of the protein  and are characterized as MAPK phosphatases. The kinase interaction motif mediates protein interactions of MAPK phosphatases, MAPK kinases (MAPKK), as well as transcription factors with the MAPKs, in animals, yeasts  and plants [7,98,99]. The mechanism of MAPK-inactivation by AP2C/MP2C is based on dephosphorylation of the pT in the MAPK activation loop pTEpY. All four AP2Cs inactivate Arabidopsis MPK6 and interact with MPK6, MPK3 or MPK4 [99–101]. Interaction between the phosphatases and the MAPKs is observed in the cytoplasm and/or in the nucleus, suggesting that in these intracellular locations the inactivation of kinases takes place [99,101]. The best-characterized B-cluster PP2Cs are AP2C1 and AP2C3, which were shown to regulate plant innate immunity and stomatal developmental pathways, respectively [99,101]. In the absence of AP2C1 and AP2C3, stomatal closing was impaired .
Different cell signalling pathways such as stress and stomatal development share the same protein components (e.g. MKK4/MKK5 and MPK3/MPK6) . Nonetheless, signal transduction leads to specific responses. Results obtained from studies on AP2Cs suggest that MAPK phosphatases could be part of the regulatory mechanisms enforcing specificity in signal transduction. For example, AP2C1 controls wound-induced MAPK activities and stress-induced ethylene responses. It also modulates plant innate immunity in response to a necrotrophic fungus . Very early, localized expression of AP2C1 at the site of wounding or pathogen attack suggests that it plays a role in plant responses to the aforementioned stress conditions . AP2C1 also suppresses MAPK activities induced by the microbe-associated molecular pattern flagellin (flg22), as well as by the damage-associated molecular pattern oligogalacturonides when expressed ectopically in Arabidopsis plants (I. Meskiene, unpublished data; ). AP2C1 negatively controls jasmonate production because ap2c1 knockout plants produce high amounts of jasmonate after wounding and show enhanced resistance to herbivores .
By contrast, AP2C3, which is closely related to AP2C1, shows a distinct expression pattern in stomata and stomatal lineage cells, which differs from those of other PP2Cs. The expression pattern and protein localization of AP2C3, as well as its ability to interact with/down-regulate the signalling activity of MAPKs, corroborates the suggestion that AP2C3 controls MPK3 and MPK6 during stomatal development . MPK3 and MPK6 inhibit stomatal development , whereas AP2C3 is able to induce conversion of almost all epidermal cells into stomata . In this process, ectopic AP2C3 expression promotes the maintenance of stomatal lineage cell proliferation and the deregulation of cell cycle genes of the early region 2/retinoblastoma 1 pathway. Inactivation of MAPKs by AP2C3 may prevent protodermal and stomatal neighbouring cells from conversion into pavement cells, leading to multiple stomata phenotype. Expression of the AP2C3 protein within its native domain (A. Schweighofer, unpublished data), as well as chemically-induced expression indicate that the protein level is a critical factor for the regulation of stomatal density. Moreover, AP2C3 nuclear localization is required for the protein phosphatase to induce cell divisions and to convert epidermal cells into stomata. The role of AP2C3 is most likely to maintain the balance between the differentiation of stomata and pavement cells on plant epidermal surfaces .
The different gene expression patterns, as well as the ability of AP2C phosphatases to act as negative regulators of the same substrate MAPKs, suggest that they may determine the specificity of the outcome of the MAPK pathway. Other signalling pathways that involve MAPK regulation by AP2C1–4 are currently under investigation.
Clade C PP2Cs: POLTERGEIST (POL) and POLTERGEIST-LIKE (PLL)
The Arabidopsis PP2C family clade C contains the phosphatases POL and PLL. Genetic studies show that POL and PLL1 act redundantly to promote stem-cell identity . pol/pll1 double mutant phenotypes show that POL and PLL1 control shoot and root meristem as well as embryo formation. POL and PLL1 are dually acetylated plasma membrane proteins that bind to the phosphatidylinositol phospholipid [PI(4)P], which activates POL in vitro .
POL/PLL1 promotes the expression of wuschel-related homeobox (WOX) transcription factors for the establishment and maintenance of the meristem (stem cells). In the shoot, POL/PLL1 control signalling downstream of CLV1 receptor to maintain the expression of the transcription factor WUSCHEL and stem cell fate . In the early embryo and root meristem, these phosphatases are potential mediators of the CLV/WUS-related CLE40/WOX5 pathway [106–108]. All pol mutants are similar to wild-type plants, although they are able to suppress meristem defects in clv1 and clv3 mutants . The observation that POL/PLL1 act downstream of a CLV receptor in establishing asymmetric stem cell divisions led to the suggestion that POL and PLL1 are responsible for the induction and/or maintenance of stem cell polarity [106,108]. It has been suggested that the polarization of POL/PLL1 protein and/or its activity leads to differential WUS expression and determines cell fate .
Signalling components of the CLV3/WUS pathway include the receptor kinase CLV1, the transmembrane kinase CRN, the receptor-like protein CLV2 and the ligand CLV3. Genetic analyses suggest that CLV3-mediated signalling inhibits POL/PLL1 .
Other PLLs, such as PLL4 and PLL5, regulate leaf development, as inferred from abnormal leaf phenotypes of pll4-1 and pll5-1 mutant plants. By contrast, no developmental role for either PLL2 or PLL3 has been observed to date .
An orthologue of Arabidopsis POL/PLL4-5, the rice PP2C XB15 regulates none-RD RLK and plant innate immunity. XB15 interacts with and dephosphorylates the RLK XA21 in vitro. Similar to POL, a XB15-GFP fusion protein resides on the plasma membrane , where the association of XB15 with XA21 RLK may take place; this is reminiscent of the RLK FLS2 and PP2C kinase-associated protein phosphatase (KAPP) interaction in Arabidopsis . The interaction of XB15 with XA21 depends on an intact XA21 juxtamembrane domain. The juxtamembrane domain contains several putative phosphorylation sites (S/ T), which suggests that phosphorylation of the juxtamembrane region and XA21 catalytic activity are important for the XB15–XA21 interaction.
The lack of or reduced expression of XB15 in rice leads to spontaneous cell death symptoms, and accounts for constitutive expression of defence-related PR genes and enhanced XA21-mediated resistance. XB15 negatively regulates the XA21-mediated defence pathway because XB15 overexpressing plants demonstrate a higher susceptibility towards Xanthomonas oryzae pv. oryzae (Xoo) harbouring the AvrXa21 activity . By contrast to Arabidopsis POL/PLLs, which regulate plant development, XB15 mutations in rice do not exhibit developmental anomalies, indicating that XB15 has distinct functions in both Arabidopsis and rice.
Clade E PP2Cs
AtPP2C6-6 (At1g03590) was identified in a yeast-two-hybrid screen as a protein interacting with Arabidopsis histone acetyltransferase GCN5 in yeast and plant cells. AtPP2C6-6 dephosphorylates GCN5 in vitro  and the regulation of GCN5 by AtPP2C6-6 is required during light-regulated gene expression for the acetylation of specific lysines on H3 and H4 histones . Although atpp2c6-6 mutant plants do not show any obvious morphological phenotype, they reveal an enhancement of histone 3 acetylation, indicating a possible negative regulation of GCN5 activity by this PP2C. AtPP2C6-6 may dephosphorylate and thereby inhibit GCN5 activity to control the activation of stress responsive genes. AtPP2C6-6 has been shown to be expressed in guard cells , suggesting that AtPP2C6-6 may represent a new component of the stomatal signalling network.
Clade F PP2C
PIA1 (PP2C induced by AvrRpm1, At2g20630) was identified in a proteomics approach as a protein up-regulated by conditional expression of the bacterial type III effector AvrRpm1 or by plant infiltration with Pseudomonas syringae carrying avrRpm1 . pia1 mutant plants are more resistant to the Pseudomonas bacterial strain Pto DC3000 (avrRpm1) but not to Pto DC3000 (avrB) or Pto DC3000. PIA1 may play a dual role in the regulation of defence-related gene expression, as it was shown to be required for both the expression and suppression of selected marker genes. PIA1 regulates the accumulation of the stress hormones ethylene and salicylic acid. This conclusion is based on the observation that pia1 mutant plants contain low levels of ethylene after wounding and salicylic acid after AvrRpm1 induction. However, it remains unclear how the opposite and mutually exclusive factors of a reduced accumulation of salicylic acid after AvrRpm1-induction and the enhanced resistance to P. syringae/avrRpm1 can be reconciled in the pia1 mutant .
The PP2C WIN2 (At4g31750) was identified in a yeast-two-hybrid screen using the bacterial effector HopW1-1 as bait . HopW1-1 is a P. syringae effector protein that causes disease resistance responses in some Arabidopsis accessions. WIN2 shows PP2C phosphatase activity in vitro. In plants, the down-regulation of WIN2 expression by RNA interference reduces HopW1-1-induced plant resistance. Ectopic expression of WIN2 enhances the resistance to Pto DC 3000 but does not influence HopW1-1-induced resistance. WIN2 levels correlate with the accumulation of salicylic acid after Pseudomonas/hopW1-1 infection . The HopW1-1/WIN2 interaction in plants remains to be determined.
DCW11 (Down-Regulated in CW-type Cytoplasmatic Male Sterility Gene 11) is orthologous to Arabidopsis clade F PP2Cs and encodes a mitochondrial PP2C, which is a down-regulated gene in CW-type cytoplasmic male sterile rice. DCW11 is expressed in mature pollen and it has been suggested to play a role in the mediation of mitochondrial signalling during pollen germination because down-regulation of DCW11 correlates with reduced seed production and the expression of the cytoplasmic male sterile marker gene AOX1a [117,118].
Protein phosphatase homologue 1 (PPH1)
The Arabidopsis PP2C phosphatase PPH1/TAP38 (Thylakoid-Associated-Phosphatase of 38 kDa) has been identified in silico as a phosphatase with a predicted chloroplast localization and shown to be localized in the thylakoid membranes [119,120]. During light acclimation processes, PPH1 affects thylakoid protein phosphorylation, which involves balancing the light-harvesting capacity of the two photosystems to maintain efficient photosynthesis in plants. Changing light conditions during photosynthesis leads to an association of the light-harvesting complex (LHC) II with photosystem I (State 2) or photosystem II (State 1). The state transitions enable rapid adaptation during short-term acclimation to changes in illumination. This acclimation requires the reversible phosphorylation of some of the LHCII proteins and involves STN7 kinase in the transition to State 2 . PPH1/TAP38 is the major LHCII phosphatase responsible for transition to State 1 [119,120]. pph1 mutant plants exhibit high levels of phosphorylated LHCII and are locked in State 2. Overexpression of PPH1 leads to reduced phosphorylation of LHCII [122,123].
KAPP interacts with different RLKs and is predicted to control different RLK-mediated signalling pathways, including meristem development via CLAVATA, plant immunity responses and/or plant hormone signalling [104,111,124]. Phenotypic and genetic studies indicate that KAPP controls plant adaptation to sodium salt stress. KAPP-deficient plants are also impaired with respect to maintaining the proper balance of cell expansion and cell division .
Recent analyses of plant PP2Cs have identified the novel regulatory modes and functions of this class of Mg2+ and Mn2+-regulated protein phosphatases. The identification of RCARs/PYR1-like proteins uncovered a new paradigm of PP2Cs, namely that PP2Cs serve as hormone co-receptors. This feature of a ligand-regulated protein phosphatase resembles PP2Bs, which are regulated by cyclophilin and the ligand cyclosporin. The regulation of MAPK activities by PP2Cs in plants indicates that protein phosphatases may act as specificity determinants in MAPK signalling. The pioneering studies conducted in plants might foster the search for similar regulatory modes of PP2C actions in animal systems.
The financial support of the Deutsche Forschungsgemeinschaft GR 938/6 is gratefully acknowledged (S.F. and E.G.). We thank Kotryna Kvederaviciute (Institute of Biotechnology, Vilnius University) for sharing unpublished data. Research is supported by the MOEL fellowship from the Austrian Research Association (ÖFG) (A.S.), the EU Mobili program (A.S. and I.M.), the Research Council of Lithuania (Project No. 039/2011) and Austrian FWF projects I-255 (EU EraNet-PG ‘PathoNet’) and L687 (I.M.). We also thank F. Assaad-Gerbert for critical reading of the manuscript.