Type 2C Protein Phosphatases Research Articles

Dephosphorylate to close: type 2C protein phosphatases regulate stomatal aperture.

Dephosphorylate to close: type 2C protein phosphatases regulate stomatal aperture.

Fine-tuning of pathogenesis-related protein 1 (PR1) activity by the melatonin biosynthetic enzyme ASMT2 in defense response to cassava bacterial blight.

Melatonin is widely involved in plant disease resistance through modulation of immune responses. Pathogenesis-related (PR) proteins play important roles in plant immune responses. However, the direct association between melatonin biosynthetic enzyme and PR protein remains elusive in plants. In this study, we found that N-acetylserotonin O-methyltransferase 2 (MeASMT2) physically interacted with MePR1 in vitro and in vivo, thereby promoting the anti-bacterial activity of MePR1 against Xanthomonas axonopodis pv. manihotis (Xam). Consistently, MeASMT2 improved the effect of MePR1 on positively regulating cassava disease resistance. In addition, we found that type 2C protein phosphatase 1 (MePP2C1) interacted with MeASMT2 to interfere with MePR1-MeASMT2 interaction, so as to inhibiting the effect of MeASMT2 and MePR1 on positively regulating cassava disease resistance. In contrast to the increased transcripts of MeASMT2 and MePR1 in response to Xam infection, the transcript of MePP2C1 was decreased upon Xam infection. Therefore, disease activated MeASMT2 was released from disease inhibited MePP2C1, so as to improving the anti-bacterial activity of MePR1, resulting in improved immune response. In summary, this study illustrates the dynamic modulation of the MePP2C1-MeASMT2-MePR1 module on cassava defense response against cassava bacterial blight (CBB), extending the understanding of the correlation between melatonin biosynthetic enzyme and PR in plants.

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase.

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

Genome-wide Identification and Evolution of the PP2C Gene Family in Eight Rosaceae Species and Expression Analysis Under Stress in Pyrus bretschneideri.

Type 2C protein phosphatase (PP2C) plays an essential role in abscisic acid (ABA) signaling transduction processes. In the current study, we identify 719 putative PP2C genes in eight Rosaceae species, including 118 in Chinese white pear, 110 in European pear, 73 in Japanese apricot, 128 in apple, 74 in peach, 65 in strawberry, 78 in sweet cherry, and 73 in black raspberry. Further, the phylogenetic analysis categorized PbrPP2C genes of Chinese white pear into twelve subgroups based on the phylogenic analysis. We observed that whole-genome duplication (WGD) and dispersed gene duplication (DSD) have expanded the Rosaceae PP2C family despite simultaneous purifying selection. Expression analysis finds that PbrPP2C genes have organ-specific functions. QRT-PCR validation of nine PbrPP2C genes of subgroup A indicates a role in ABA-mediated response to abiotic stress. Finally, we find that five PbrPP2C genes of subgroup A function in the nucleus. In summary, our research suggests that the PP2C family functions to modulate ABA signals and responds to abiotic stress.

Downregulation of lncRNA PpL-T31511 and Pp-miRn182 Promotes Hydrogen Cyanamide-Induced Endodormancy Release through the PP2C-H2O2 Pathway in Pear (Pyrus pyrifolia).

Bud endodormancy is an important, complex process subject to both genetic and epigenetic control, the mechanism of which is still unclear. The endogenous hormone abscisic acid (ABA) and its signaling pathway play important roles in the endodormancy process, in which the type 2C protein phosphatases (PP2Cs) is key to the ABA signal pathway. Due to its excellent effect on endodormancy release, hydrogen cyanamide (HC) treatment is considered an effective measure to study the mechanism of endodormancy release. In this study, RNA-Seq analysis was conducted on endodormant floral buds of pear (Pyrus pyrifolia) with HC treatment, and the HC-induced PP2C gene PpPP2C1 was identified. Next, software prediction, expression tests and transient assays revealed that lncRNA PpL-T31511-derived Pp-miRn182 targets PpPP2C1. The expression analysis showed that HC treatment upregulated the expression of PpPP2C1 and downregulated the expression of PpL-T31511 and Pp-miRn182. Moreover, HC treatment inhibited the accumulation of ABA signaling pathway-related genes and hydrogen peroxide (H2O2). Furthermore, overexpression of Pp-miRn182 reduced the inhibitory effect of PpPP2C1 on the H2O2 content. In summary, our study suggests that downregulation of PpL-T31511-derived Pp-miRn182 promotes HC-induced endodormancy release in pear plants through the PP2C-H2O2 pathway.

A type-2C protein phosphatase (GhDRP1) participates in cotton (Gossypium hirsutum) response to drought stress.

GhDRP1 acts as a negatively regulator to participate in response to drought stress possibly by modulating ABA signaling pathway and flavonoid biosynthesis pathway which affects stomata movement and thus water loss, ROS scavenging enzymes, and proline accumulation in cotton. Type-2C protein phosphatases (PP2C) may play important roles in plant stress signal transduction. Here, we show the evidence that a cotton PP2C protein GhDRP1 participates in plant response to drought stress. GhDRP1 gene encodes an active type-2C protein phosphatase (PP2C) and its expression is significantly induced in cotton by drought stress. Compared with wild type, the GhDRP1 overexpression (OE) transgenic cotton and Arabidopsis displayed reduced drought tolerance, whereas GhDRP1-silenced (RNAi) cotton showed enhanced drought tolerance. Under drought stress, malondialdehyde content was lower, whereas superoxide dismutase and peroxidase activities, proline content, stomata closure and relative water content were higher in GhDRP1 RNAi plants compared with those in wild type. In contrast, GhDRP1 OE plants showed the opposite phenotype under the same conditions. Expression levels of some stress-related and flavonoid biosynthesis-related genes were altered in GhDRP1 transgenic plants under drought stress. Additionally, GhDRP1 protein could interact with other proteins such as PYLs, SNF1-related protein kinase and GLK1-like protein. Collectively, these data suggest that GhDRP1 participates in plant response to drought stress possibly by modulating ABA signaling pathway and flavonoid biosynthesis pathway which affects stomata movement and thus water loss, ROS scavenging enzymes, and proline accumulation in cotton.

New Abscisic Acid Derivatives Revealed Adequate Regulation of Stomatal, Transcriptional, and Developmental Responses to Conquer Drought.

The phytohormone abscisic acid (ABA) plays an important role in plant stress response, mainly against desiccation. Hence, ABA receptor agonists may function as agents to enhance drought tolerance in crops. ABA exhibits diverse functions that impact plant development and are regulated by various ABA receptor subfamilies. Indeed, we previously reported that 3'-alkyl ABAs exhibit diverse receptor specificities and that 3'-butyl ABA induced a drought stress response without eliciting growth inhibitory effects in Arabidopsis seedlings. Thus, to further investigate plant responses induced by 3'-butyl ABA, as well as the receptors that control the opposing stress and growth responses, we designed new 3'-alkyl ABA derivatives. In addition to the 3'-alkyl chain, a cyclopropyl group was attached to position 3 of ABA to occupy the C6 cleft in the ABA-binding pocket of the receptors, which served to increase the binding affinity and specificity to a certain receptor set. Additionally, the inhibitory activity of pyrabactin resistance 1 (PYR1) and PYR1-like (PYL1) proteins against type 2C protein phosphatase increased following incorporation of the 3-cyclopropyl group in all tested 3'-alkyl ABAs. Interestingly, 3'-butyl ABA induced the highest tolerance against drought stress, compared with 3-cyclopropyl derivatives. To investigate the molecular mechanism underlying the effects elicited by different chemical treatments, those of ABA derivatives on stomatal closure, growth, and gene expression were studied. Evaluation of the receptors activated by ABA derivatives and the plant responses revealed the induction of PYR1, PYL1, PYL2, and PYL5, mediated stomatal closure, and regulated transcription, consequently leading to drought tolerance in plants.

CaADIP1‐dependent CaADIK1‐kinase activation is required for abscisic acid signalling and drought stress response in Capsicum annuum

Induction of the abscisic acid (ABA) signalling network is associated with various stress conditions, including cold, high salinity and drought. As core ABA signalling components, group A type 2C protein phosphatases (PP2Cs) interact with and inhibit snf1-related protein kinase2s. Here, we isolated and characterised the pepper mitogen-activated protein kinase kinase kinase CaADIK1, which interacts with the group A PP2C CaADIP1. CaADIK1 transcripts were induced by abiotic stresses, and CaADIK1 localised in the nucleus and cytoplasm. We verified that CaADIP1 inhibits the autokinase activity of CaADIK1; moreover, the kinase activity of CaADIK1 is enhanced by drought stress. We performed genetic analysis using CaADIK1-silenced pepper and CaADIK1-overexpressing (OX) Arabidopsis plants. CaADIK1-silenced pepper plants showed drought-sensitive phenotypes, whereas CaADIK1-OX Arabidopsis plants showed ABA-sensitive and drought-tolerant phenotypes. In CaADIK1K32N -OX Arabidopsis plants mutated at the ATP-binding site, the ABA-insensitive and drought-sensitive phenotypes were restored. Taken together, our findings show that CaADIK1 positively regulates the ABA-dependent drought stress response and is inhibited by CaADIP1.

TMK4 receptor kinase negatively modulates ABA signaling by phosphorylating ABI2 and enhancing its activity.

In plants, clade A type 2C protein phosphatases (PP2CAs) have emerged as major players in abscisic acid (ABA)-regulated stress responses by inhibiting protein kinase activity. However, how different internal and external environmental signals modulate the activity of PP2CAs are not well known. The transmembrane kinase (TMK) protein 4 (TMK4), one member of a previously identified receptor kinase subfamily on the plasma membrane that plays vital roles in plant cell growth, directly interacts with PP2CAs member (ABA-Insensitive 2, ABI2). tmk4 mutant is hypersensitive to ABA in both ABA-inhibited seed germination and primary root growth, indicating that TMK4 is a negative regulator in ABA signaling pathway. Further analyses indicate that TMK4 phosphorylates ABI2 at three conserved Ser residues, thus enhancing the activity of ABI2. The phosphorylation-mimic ABI2S139DS140DS266D can complement but non-phosphorylated form ABI2S139AS140AS266A cannot complement ABA hypersensitive phenotype of the loss-of-function mutant abi1-2abi2-2. This study provides a previously unidentified mechanism for positively regulating ABI2 by a plasma membrane protein kinase.

Stomatal regulation: Role of H2S-induced persulfidation in ABA signaling

Stomatal regulation: Role of H2S-induced persulfidation in ABA signaling

Pepper E3 ligase CaAIRE1 promotes ABA sensitivity and drought tolerance by degradation of protein phosphatase CaAITP1.

Plants have developed defense mechanisms to survive in extreme environmental conditions. Abscisic acid (ABA) is a key phytohormone associated with plant adaptation to environmental stress. In this study, we isolated and functionally characterized the pepper RING-type E3 ligase CaAIRE1 (Capsicum annuum ABA Induced RING-type E3 ligase 1) containing the C3HC4-type RING domain. CaAIRE1 was induced by ABA and drought, and CaAIRE1 had E3 ligase activity. CaAIRE1-silenced pepper and CaAIRE1-overexpressing Arabidopsis presented drought-sensitive and drought-tolerant phenotypes, respectively, which were accompanied by altered transpiration water loss and ABA sensitivity. Moreover, we found that CaAIRE1 interacts with and ubiquitinates the pepper type 2C protein phosphatase, CaAITP1 (Capsicum annuum CaAIRE1 Interacting Target Phosphatase 1). A cell-free degradation assay with CaAIRE1-silenced peppers and CaAIRE1-overexpressing Arabidopsis plants revealed that the CaAITP1 protein level was negatively modulated by the expression level of CaAIRE1. In contrast to CaAIRE1, CaAITP1-silenced pepper showed ABA-sensitivity phenotypes. CaAITP1-overexpressing Arabidopsis plants were the most insensitive phenotypes to ABA compared with the wild type and other pepper PP2C-overexpressing plants. Taken together, our data indicate that CaAITP1 plays a major role as a negative modulator in ABA signaling, and CaAIRE1 regulates the ABA signaling and drought response through modulation of CaAITP1 stability.

Potent ABA-independent activation of engineered PYL3.

Abscisic acid (ABA) plays a vital role in many developmental processes and the response to adaptive stress in plants. Under drought stress, plants enhance levels of ABA and activate ABA receptors, but under harsh environmental stress, plants usually cannot efficiently synthesize and release sufficient quantities of ABA. The response of plants to harsh environmental stress may be improved through ABA‐independent activation of ABA receptors. The molecular basis of ABA‐independent inhibition of group A protein phosphatases type 2C (PP2Cs) by pyrabactin resistance/Pyr1‐like (PYR1/PYLs) is not yet clear. Here, we used our previously reported structures of PYL3 to first obtain the monomeric PYL3 mutant and then to introduce bulky hydrophobic residue substitutions to promote the closure of the Gate/L6/CL2 loop, thereby mimicking the conformation of ABA occupancy. Through structure‐guided mutagenesis and biochemical characterization, we investigated the mechanism of ABA‐independent activation of PYL3. Two types of PYL3 mutants were obtained: (a) PYL3 V108K V107L V192F can bind to ABA and effectively inhibit HAB1 without ABA; (b) PYL3 V108K V107F V192F, PYL3 V108K V107L V192F L111F and PYL3 V108K V107F V192F L111F cannot recognize ABA but can greatly inhibit HAB1 without ABA. Intriguingly, the ability of PYL3 mutants to bind to ABA was severely compromised if any two of three variable residues (V107, V192 and L111) were mutated into a bulky hydrophobic residue. The introduction of PYL3 mutants into transgenic plants will help elucidate the functionality of PYL3 in vivo and may facilitate the future production of transgenic crops with high yield and tolerance of abiotic stresses.

Protein Phosphatase Type 2C Functions in Phytohormone-Dependent Pathways and in Plant Responses to Abiotic Stresses.

Plants, as sessile organisms, are susceptible to a myriad of stress factors, especially abiotic stresses. Over the course of evolution, they have developed multiple mechanisms to sense and transduce environmental stimuli for appropriate responses. Among those, phosphorylation and dephosphorylation, regulated by protein kinases and protein phosphatases, respectively, are considered crucial signal transduction mechanisms. Regarding the latter group, protein phosphatases type 2C (PP2Cs) represent the largest division of PPs. In addition, the discovery of regulatory functions of PP2Cs in the abscisic acid (ABA)-signaling pathway, the major signal transduction pathway in abiotic stress responses, indicates the significant importance of PP2C members in plant adaptation to adverse environmental factors. In this review, current understanding of the roles of PP2Cs in different phytohormone-dependent pathways related to abiotic stress is summarized, highlighting the crosstalk between the ABA-signaling pathway with other hormonal pathways via certain ABA-related PP2Cs. We also updated the progress of in planta characterization studies of PP2Cs under abiotic stress conditions, providing knowledge of PP2C manipulation in developing abiotic stress-tolerant crops.

Identification of the PP2C gene family in paper mulberry (Broussonetia papyrifera) and its roles in the regulation mechanism of the response to cold stress.

To study the possible roles of type-2C protein phosphatases (PP2Cs) which have been confirmed to play roles in the response to diverse abiotic stresses in paper mulberry, we launched a series of genomic and functional studies of BpPP2Cs. Sixty-three PP2C proteins in paper mulberry (Broussonetia papyrifera) were classified into 13 clades. Four BpPP2Cs with kinase domains were verified to be highly conserved in organisms ranging from algae to dicots. Seven pairs of BpPP2C genes were found to be expanding, and 18 BpPP2C genes had orthologues in Arabidopsis. BpPP2Cs showed broad expression in different tissues; the expression levels of 18 BpPP2Cs were changed and the phosphorylation levels of seven BpPP2C proteins increased at low temperature. Cold-response elements were found in the promoter region of 31 BpPP2Cs. Finally, Bp01g0320 was found to act as a hub protein and Bp01g0512 and Bp09g1278 played key roles in the ABA-signaling pathway and MAPK cascades, respectively. These results suggest that the PP2C gene family of paper mulberry is evolutionarily conserved and participates the regulation of the response to cold stress, which will play a vital role in further research on phosphatases in paper mulberry.

A type 2C protein phosphatase activates high-affinity nitrate uptake by dephosphorylating NRT2.1.

The nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation1,2. However, the critical enzymes required for the post-translational activation of NRT2.1 remain to be identified. Here, we show that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating Ser501 of NRT2.1, a residue that functions as a negative phospho-switch in Arabidopsis2. CEPH is predominantly expressed in epidermal and cortex cells in roots and is upregulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signalling from shoots3,4. The loss of CEPH leads to marked decreases in high-affinity nitrate uptake, tissue nitrate content and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in the N-starvation-dependent activation of NRT2.1 and provide molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment.

Transcriptome analysis of salt stress responsiveness in the seedlings of wild and cultivated Ricinus communis L

Soil salinity is one of the major environmental factors, influencing agricultural productivity of crops. As a non-edible and ideal oilseed crop, castor (Ricinus communis L.) has great industrial value in biofuel, but molecular mechanisms of salt stress regulation are still unknown. In this study, the differentially expressed genes (DEGs) for differential salt tolerance in two castor cultivar (wild castor : Y, cultivated castor ‘Tongbi 5’: Z) were identified. 12 libraries were sampled for Illumina high-throughput sequencing to consider 132,426 nonredundant unigenes and 31,221 gene loci. Multiple phytohormones and transcription factors (TFs) were correlated with salt-tolerance and differently enriched in these two genotypes. The type 2C protein phosphatases (PP2C) homologs were all upregulated under salt stress. Importantly, IAA (1), DELLA (1) and Jasmonate zim domain (JAZ) (1) were also identified and found to be differentially expressed. Based on the co-expressed module by regulatory networks and heatmap analysis, ERF/AP2, WRKY and bHLH families were prominently participate in high salt stress response of wild and cultivated castor. Finally, these results highlight that the hub DEGs and families were more accumulated in cultivated castor than those in wild castor, providing novel insights into the salinity adaptive mechanisms and genetic improvement in castor.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways.

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

Transcriptional Regulation of Protein Phosphatase 2C Genes to Modulate Abscisic Acid Signaling.

The plant hormone abscisic acid (ABA) triggers cellular tolerance responses to osmotic stress caused by drought and salinity. ABA controls the turgor pressure of guard cells in the plant epidermis, leading to stomatal closure to minimize water loss. However, stomatal apertures open to uptake CO2 for photosynthesis even under stress conditions. ABA modulates its signaling pathway via negative feedback regulation to maintain plant homeostasis. In the nuclei of guard cells, the clade A type 2C protein phosphatases (PP2Cs) counteract SnRK2 kinases by physical interaction, and thereby inhibit activation of the transcription factors that mediate ABA-responsive gene expression. Under osmotic stress conditions, PP2Cs bind to soluble ABA receptors to capture ABA and release active SnRK2s. Thus, PP2Cs function as a switch at the center of the ABA signaling network. ABA induces the expression of genes encoding repressors or activators of PP2C gene transcription. These regulators mediate the conversion of PP2C chromatins from a repressive to an active state for gene transcription. The stress-induced chromatin remodeling states of ABA-responsive genes could be memorized and transmitted to plant progeny; i.e., transgenerational epigenetic inheritance. This review focuses on the mechanism by which PP2C gene transcription modulates ABA signaling.

CORRECTION: ABI1 and PP2CA phosphatases are negative regulators of Snf1-related protein kinase1 signaling in Arabidopsis.

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways.

Phosphatase OsPP2C27 directly dephosphorylates OsMAPK3 and OsbHLH002 to negatively regulate cold tolerance in rice.

Improving chilling tolerance is a major target of rice breeding. The OsMAPK3-OsbHLH002-OsTPP1 signalling pathway enhances chilling tolerance in rice: the kinase is activated by cold stress, and subsequently the transcription factor is phosphorylated by the activated kinase, triggering the expression of cold response genes. However, it is largely unknown how this pathway is suppressed in time to avoid it being in a continuously activated state. We found that a novel type 2C protein phosphatase, OsPP2C27, functions as a negative regulator of the OsMAPK3-OsbHLH002-OsTPP1 pathway. A dynamic change in OsMAPK3 activity was found during cold treatment. We show that OsPP2C27 interacts physically with and dephosphorylates OsMAPK3 in vitro and in vivo. Interestingly, OsPP2C27 can also directly dephosphorylate OsbHLH002, the target of OsMAPK3. After cold treatment, survival rates were higher in OsPP2C27-RNAi lines and a T-DNA insertion mutant, and lower in OsPP2C27-overexpression lines, compared to wild type. Moreover, expression of the OsTPP1 and OsDREBs were increased in OsPP2C27-RNAi lines and decreased in OsPP2C27-overexpression lines. These results indicate that cold-induced OsPP2C27 negatively regulates the OsMAPK3-OsbHLH002-OsTPP1 signalling pathway by directly dephosphorylating both phospho-OsMAPK3 and phospho-OsbHLH002, preventing the sustained activation of a positive pathway for cold stress and maintaining normal growth under chilling conditions.