PP2C Genes Research Articles

Characteristics of PP2C Gene Family in Foxtail Millet ( Setaria italica )

PP2Cs are a group of monomeric serine/threonine protein phosphatases, which play an important role in regulation of hormone signaling pathways such as abscisic acid(ABA), jasmonic acid(JA) and salicylic acid(SA) in eukaryotes. In this study,we identified 80 candidate PP2C genes from foxtail millet genome via sequence alignment, and cluster analysis showed that PP2Cs in foxtail millet were divided into 12 subfamilies(subfamily A, B, C, D, E1, E2, F1, F2, G, H, I, and J). Foxtail millet and Arabidopsis share A, B, C, D, E1, E2, F1, F2, G, H, and I, while subfamily J is only in foxtail millet and subfamily L is only in Arabidopsis. Ten members of subfamily A in foxtail millet were named as SiPP2CA1–10, and gene expression profiles showed that the expression of these genes was induced by ABA, drought, high salt, cold and low nitrogen stresses. Among them,SiPP2CA6 and SiPP2CA8 showed high expression level in all treatments. Promoter analysis identified a variety of cis-acting elements involved in stress responses in promoter region of members in subfamily A, and a specific element responding to low nitrogen stress in SiPP2CA5, SiPP2CA6, SiPP2CA7, and SiPP2CA8. Further study demonstrated that SiPP2CA8 mainly expressed in root, and its expression remained at high level under low nitrogen stress. Subcellular localization showed that SiPP2CA8 was localized in cytomembrane, cytoplasm and nucleus. Bimolecular fluorescence complementation(BiFC) assay demonstrated that SiPP2CA8 interacted with an ABA receptor like protein, SiRCAR3(gene locus Si018317m.g), in cytomembrane, cytoplasm and nucleus. These results suggested that SiPP2CA8 may participate in ABA signaling pathway in foxtail millet.

Mithochondrial type 2C protein phosphatases CaPtc5p, CaPtc6p, and CaPtc7p play vital roles in cellular responses to antifungal drugs and cadmium inCandida albicans

Type 2C protein phosphatases (PP2C) are monomeric enzymes that require magnesium or manganese ions for their activities. There are seven PP2C genes in Candida albicans. Here, we demonstrate that CaPTC5 encodes a mitochondrial PP2C enzyme. Expression of CaPTC5 transcripts remains constant during the serum-induced hyphal development. Deletion of CaPTC5 does not affect the in vitro filamentation but renders C. albicans cells sensitive to terbinafine and cadmium, and this sensitivity is complemented by the Saccharomyces cerevisiae ScPTC5. Deletion of CaPTC6 does not have any additive effect on, but deletion of CaPTC7 blocks, the terbinafine sensitivity owing to deletion of CaPTC5. In addition, we have shown that deletion of CaPTC6 also renders C. albicans cells sensitive to cadmium, while deletion of CaPTC7 leads to a high cadmium tolerance, and this tolerance is abolished by further deletion of CaPTC5 or CaPTC6. Furthermore, double deletion of CaPTC6 and CaPTC7 renders C. albicans cells more tolerant to azoles, but deletion of CaPTC5 and CaPTC7 slightly increases the azole sensitivity of C. albicans cells. Our results demonstrate that three mitochondrial PP2C genes CaPTC5, CaPTC6 and CaPTC7 interact differentially in the response of C. albicans cells to antifungal drugs and cadmium.

A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum.

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.

Isolation of a novel PP2C gene from rice and its response to abiotic stresses

Biotic and abiotic stresses adversely affect plant growth and improvement. In order to discover new stress-tolerance genes in rice ( Oryza sativa L.), we analyzed a global genome expression profiling of the indica cultivar Pei’ai 64S, using Affymetrix rice expression chip, subjected to cold, drought or heat stress. A large number of genes highly up regulated or down regulated were identified under the stresses. One of these genes, O. sativa L. protein phosphatase2C-l (OsPP2C1), was highly induced in leaf and panicle at the heading and flowering stages, in response to all stresses. The expression profile of OsPP2C1 obtained by the microarray analysis was confirmed by quantitative real-time PCR (qRTPCR). The two sets of data matched very well, suggesting that OsPP2C1 is a multiple stresses responsive gene in rice. In order to study its function in stress tolerance, the cDNA of the gene was cloned by reverse transcription (RT-PCR). Sequence analysis showed that there is 3bp discrepancy between the cDNA of japonica and indica group. Deduced protein from OsPP2C1 cDNA has molecular weight about 37.6 kD and isoelectric points about 6.16. Analysis of the putative promoter regions showed that there were about 10 kinds of stress induced by cis-acting elements. Based on the mentioned analyses and results obtained, we propose that OsPP2C1 is a novel candidate gene involved in stress tolerance in rice. Key words: Rice, stress, microarray, real-time PCR, analysis function.

Promoter activation by ACGT in response to salicylic and abscisic acids is differentially regulated by the spacing between two copies of the motif

A variety of small sequence motifs, located upstream of minimal promoters influence gene expression. The function of the core sequence ACGT, present in a family of commonly occurring cis acting transcription regulatory motifs was investigated in the background of an artificially designed synthetic promoter sequence. The ACGT was placed in one or two copies and separated by different spacer lengths between the two copies, to study their affect on the expression of basal promoter in plant cells. The activation of transcription by the ACGT element was examined by transient and stable transformation in tobacco, using gusA as the reporter gene. The analysis shows that the expression of the reporter gene was influenced differently by spacing between two adjacent copies of the motif. Two copies of the ACGT element separated by 5 nucleotides gave highest activation. This configuration imparted salicylic acid inducibility to the basal promoter. However, two copies of ACGT separated by 25 nucleotides allowed the promoter to be induced by abscisic acid but not salicylic acid. Computational analysis of the Arabidopsis thaliana genome database showed the presence of these motifs in several genes associated with a variety of stress responses. The results on motif-related inducibility by salicylic acid and abscisic acid, as seen in a synthetic sequence background were validated by experiments on the expression of the native promoter of a protein phosphatase 2C-like gene of Arabidopsis in tobacco leaves. The study supports the importance of spacing between the ACGT sequence motifs on the elicitor specific modulation of gene expression and demonstrates the role of different ACGT motifs in the regulation of PP2C gene expression.

Candida albicans CaPTC6 is a functional homologue for Saccharomyces cerevisiae ScPTC6 and encodes a type 2C protein phosphatase

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6-encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast-form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology.

Identification and characterization of the type 2C protein phosphatase Ptc4p in the human fungal pathogen Candida albicans

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, named from PTC1 to PTC7, in Saccharomyces cerevisiae. In the current study we identified the CaPTC4 gene in Candida albicans and demonstrated that the CaPtc4p protein is a typical PP2C enzyme, which is highly conserved in fungal species. Deletion of CaPTC4 renders Candida cells sensitive to sodium and potassium ions as well as to antifungal azole drugs. In addition, we have shown that CaPtc4p is localized in the mitochondrion, suggesting that CaPtc4p is likely to be involved in the regulation of a mitochondrial function related to ion homeostasis.

AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation

A myriad of drought stress-inducible genes have been reported, and many of these are activated by abscisic acid (ABA). In the promoter regions of such ABA-regulated genes, conserved cis-elements, designated ABA-responsive elements (ABREs), control gene expression via bZIP-type AREB/ABF transcription factors. Although all three members of the AREB/ABF subfamily, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress, it remains unclear whether these are functional homologs. Here, we report that all three AREB/ABF transcription factors require ABA for full activation, can form hetero- or homodimers to function in nuclei, and can interact with SRK2D/SnRK2.2, an SnRK2 protein kinase that was identified as a regulator of AREB1. Along with the tissue-specific expression patterns of these genes and the subcellular localization of their encoded proteins, these findings clearly indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. To elucidate the role of these AREB/ABF transcription factors, we generated an areb1 areb2 abf3 triple mutant. Large-scale transcriptome analysis, which showed that stress-responsive gene expression is remarkably impaired in the triple mutant, revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class and group-Ab PP2C genes and transcription factors. The areb1 areb2 abf3 triple mutant is more resistant to ABA than are the other single and double mutants with respect to primary root growth, and it displays reduced drought tolerance. Thus, these results indicate that AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent gene expression for ABA signaling under conditions of water stress.

Differential Expression of Serine/Threonine Protein Phosphatase Type-2C Under Drought Stress in Maize

Differential display reverse transcription polymerase chain reaction was used to study the messenger RNA differential expression of a drought tolerant inbred line ‘81565’ under drought stress and well-watered control. Fragments of MD1, MD2, and MD3 were found to be differentially expressed under drought stress. While the expression of MD1 and MD2 was down-regulated, the expression of MD3 was up-regulated. Sequence analysis of the three fragments revealed that MD1 shared 97% nucleotide sequence identity with maize chloroplast matK, a gene encoding RNA maturase involved in group II intron splicing of RNA transcript; MD2 had 99% identity with PP2C, a gene encoding serine/threonine phosphorylase 2C in resurrection grass Sporobolus stapfianus; and MD3 exhibited 99% sequence identity with a rice gene encoding metacaspase, an arginine/lysine-specific cysteine protease. Based on MD2 fragment sequence, a full-length complementary DNA of 1,731 bp was isolated. An open reading frame of 1,167 bp was found and predicted to encode a protein of 388 amino acids, containing the catalytic domain and conserved residues of serine/threonine protein phosphatase 2C. This open reading frame was identified as a new member of the PP2C gene family in maize and designated as ZmPP2Ca. The differential expression of ZmPP2Ca and enzyme activity of PP2C were also investigated in three drought-tolerant and two sensitive maize inbred lines under drought stress and well-watered (control) conditions by real-time fluorescence quantitative PCR and a non-radioactive labeled method, respectively. Down-regulation of the ZmPP2Ca transcript was observed in all three drought tolerant lines, whereas transcript levels were up-regulated in the two sensitive lines under drought stress. In light of the importance of protein kinases in signal transduction pathways, it is proposed that ZmPP2Ca may play a role in the signal transduction of maize in response to drought stress.

Role of Type 2C Protein Phosphatases in Growth Regulation and in Cellular Stress Signaling

ABSTRACTA number of interesting features, phenotypes, and potential clinical applications have recently been ascribed to the type 2C family of protein phosphatases. Thus far, 16 different PP2C genes have been identified in the human genome, encoding (by means of alternative splicing) for at least 22 different isozymes. Virtually ever since their discovery, type 2C phosphatases have been predominantly linked to cell growth and to cellular stress signaling. Here, we provide an overview of the involvement of type 2C phosphatases in these two processes, and we show that four of them (PP2Cα, PP2Cβ, ILKAP, and PHLPP) can be expected to function as tumor suppressor proteins, and one as an oncoprotein (PP2Cδ /Wip1). In addition, we demonstrate that in virtually all cases in which they have been linked to the stress response, PP2Cs act as inhibitors of cellular stress signaling. Based on the vast amount of experimental evidence obtained thus far, it therefore seems justified to conclude that type 2C protein phosphatases are important physiological regulators of cell growth and of cellular stress signaling.

Transcriptional Profiling of the Protein Phosphatase 2C Family in Yeast Provides Insights into the Unique Functional Roles of Ptc1

Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes.

塩生植物アイスプラントのタンパク質脱リン酸化酵素タイプ2C（PP2C）遺伝子を導入したシロイヌナズナの解析

塩生植物アイスプラントのタンパク質脱リン酸化酵素タイプ2C（PP2C）遺伝子を導入したシロイヌナズナの解析

The YHR076w gene encodes a type 2C protein phosphatase and represents the seventh PP2C gene in budding yeast

The YHR076w gene encodes a type 2C protein phosphatase and represents the seventh PP2C gene in budding yeast

Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana.

Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The cold and drought-induced expression of these genes was ABA-dependent, but divergent in various ABA signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue-specific expression as well as in temporal induction in response to low temperature. To elucidate the function of AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition. Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in antisense plants. These plants displayed increased sensitivity to ABA both during development of frost tolerance and during seed germination, but not in their drought responses. Furthermore, the expression of cold-and ABA-induced genes was enhanced in transgenic antisense plants. Our results suggest that AtPP2CA is a negative regulator of ABA responses during cold acclimation.

Alternative promoters direct tissue-specific expression of the mouse protein phosphatase 2Cbeta gene.

Type 2C protein phosphatases (PP2Cs), a class of ubiquitous and evolutionally conserved serine/threonine protein phosphatases, are encoded in at least four distinct genes and implicated in the regulation of various cellular functions. Of these four PP2C genes, the expression of the PP2Cbeta gene has been reported to be tissue-specific and development-dependent. To understand more precisely the regulatory mechanism of this expression, we have isolated and characterized overlapping mouse genomic lambda clones. A comparison of genomic sequences with PP2Cbeta cDNA sequences provided information on the structure and localization of intron/exon boundaries and indicated that PP2Cbeta isoforms with different 5' termini were generated by alternative splicing of its pre-mRNA. The 5'-flanking region of exon 1 had features characteristic of a housekeeping gene: it was GC-rich, lacked TATA boxes and CAAT boxes in the standard positions, and contained potential binding sites for the transcription factor SP1. In the 5'-flanking region of exon 2, several consensus sequences were found, such as a TATA-like sequence and negative regulatory element box-1, -2 and -3. Subsequent analysis by transient transfection assay with a reporter gene showed that these regions act as distinct promoters. Analysis of PP2Cbeta transcripts by reverse transcriptase-PCR showed that exon-1 transcripts were expressed ubiquitously in all of the tissues examined, whereas exon-2 transcripts were predominantly expressed in the testis, intestine and liver. These results suggest that the alternative usage of two promoters within the PP2Cbeta gene regulates tissue-specific expression of PP2Cbeta mRNA.