Genome-wide analysis of the PYL-PP2C-SnRK2s family in the ABA signaling pathway of pitaya reveals its expression profiles under canker disease stress.

BACKGROUND: Abscisic acid (ABA) plays a crucial role in seed dormancy, germination, and growth, as well as in regulating plant responses to environmental stresses during plant growth and development. However, detailed information about the PYL-PP2C-SnRK2s family, a central component of the ABA signaling pathway, is not known in pitaya. RESULTS: In this study, we identified 19 pyrabactin resistance-likes (PYLs), 70 type 2 C protein phosphatases (PP2Cs), and 14 SNF1-related protein kinase 2s (SnRK2s) from pitaya. In pitaya, tandem duplication was the primary mechanism for amplifying the PYL-PP2C-SnRK2s family. Co-linearity analysis revealed more homologous PYL-PP2C-SnRK2s gene pairs located in collinear blocks between pitaya and Beta vulgaris L. than that between pitaya and Arabidopsis. Transcriptome analysis showed that the PYL-PP2C-SnRK2s gene family plays a role in pitaya's response to infection by N. dimidiatum. By spraying ABA on pitaya and subsequently inoculating it with N. dimidiatum, we conducted qRT-PCR experiments to observe the response of the PYL-PP2C-SnRK2s gene family and disease resistance-related genes to ABA. These treatments significantly enhanced pitaya's resistance to pitaya canker. Further protein interaction network analysis helped us identify five key PYLs genes that were upregulated during the interaction between pitaya and N. dimidiatum, and their expression patterns were verified by qRT-PCR. Subcellular localization analysis revealed that the PYL (Hp1879) gene is primarily distributed in the nucleus. CONCLUSION: This study enhances our understanding of the response of PYL-PP2C-SnRK2s to ABA and also offers a new perspective on pitaya disease resistance.

Drought Responses in Poaceae: Exploring the Core Components of the ABA Signaling Pathway in Setaria italica and Setaria viridis.

Drought severely impacts plant development and reproduction, reducing biomass and seed number, and altering flowering patterns. Drought-tolerant Setaria italica and Setaria viridis species have emerged as prominent model species for investigating water deficit responses in the Poaceae family, the most important source of food and biofuel biomass worldwide. In higher plants, abscisic acid (ABA) regulates environmental stress responses, and its signaling entails interactions between PYR/PYL/RCAR receptors and clade A PP2C phosphatases, which in turn modulate SnRK2 kinases via reversible phosphorylation to activate ABA-responsive genes. To compare the diversity of PYR/PYL/RCAR, PP2C, and SnRK2 between S. italica and S. viridis, and their involvement in water deficit responses, we examined gene and regulatory region structures, investigated orthology relationships, and analyzed their gene expression patterns under water stress via a meta-analysis approach. Results showed that coding and regulatory sequences of PYR/PYL/RCARs, PP2Cs, and SnRK2s are highly conserved between Setaria spp., allowing us to propose pairs of orthologous genes for all the loci identified. Phylogenetic relationships indicate which clades of Setaria spp. sequences are homologous to the functionally well-characterized Arabidopsis thaliana PYR/PYL/RCAR, PP2C, and SnRK2 genes. Gene expression analysis showed a general downregulation of PYL genes, contrasting with upregulation of PP2C genes, and variable expression modulation of SnRK2 genes under drought stress. This complex network implies that ABA core signaling is a diverse and multifaceted process. Through our analysis, we identified promising candidate genes for further functional characterization, with great potential as targets for drought resistance studies, ultimately leading to advances in Poaceae biology and crop-breeding strategies.

Identification of PP2Cs in six rosaceae species highlights RcPP2C24 as a negative regulator in rose drought tolerance.

Drought is a major environmental stress that limits plant growth, so it's important to identify drought-responsive genes to understand the mechanism of drought response and breed drought-tolerant roses. Protein phosphatase 2C (PP2C) plays a crucial role in plant abiotic stress response. In this study, we identified 412 putative PP2Cs from six Rosaceae species. These genes were divided into twelve clades, with clade A containing the largest number of PP2Cs (14.1%). Clade A PP2Cs are known for their important role in ABA-mediated drought stress response; therefore, the analysis focused on these specific genes. Conserved motif analysis revealed that clade A PP2Cs in these six Rosaceae species shared conserved C-terminal catalytic domains. Collinearity analysis indicated that segmental duplication events played a significant role in the evolution of clade A PP2Cs in Rosaceae. Analysis of the expression of 11 clade A RcPP2Cs showed that approximately 60% of these genes responded to drought, high temperature, and salt stress. Among them, RcPP2C24 exhibited the highest responsiveness to both drought and ABA. Furthermore, overexpression of RcPP2C24 significantly reduced drought tolerance in transgenic tobacco by increasing stomatal aperture after exposure to drought stress. The transient overexpression of RcPP2C24 weakened the dehydration tolerance of rose petal discs, while its silencing increased their dehydration tolerance. In summary, our study identified PP2Cs in six Rosaceae species and highlighted the negative role of RcPP2C24 on rose's drought tolerance by inhibiting stomatal closure. Our findings provide valuable insights into understanding the mechanism behind rose's response to drought.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Genome-wide analysis and expression pattern of the ZoPP2C gene family in Zingiber officinale Roscoe.

BACKGROUND: Protein phosphatases type 2C (PP2C) are heavily involved in plant growth and development, hormone-related signaling pathways and the response of various biotic and abiotic stresses. However, a comprehensive report identifying the genome-scale of PP2C gene family in ginger is yet to be published. RESULTS: In this study, 97 ZoPP2C genes were identified based on the ginger genome. These genes were classified into 15 branches (A-O) according to the phylogenetic analysis and distributed unevenly on 11 ginger chromosomes. The proteins mainly functioned in the nucleus. Similar motif patterns and exon/intron arrangement structures were identified in the same subfamily of ZoPP2Cs. Collinearity analysis indicated that ZoPP2Cs had 33 pairs of fragment duplicated events uniformly distributed on the corresponding chromosomes. Furthermore, ZoPP2Cs showed greater evolutionary proximity to banana's PP2Cs. The forecast of cis-regulatory elements and transcription factor binding sites demonstrated that ZoPP2Cs participate in ginger growth, development, and responses to hormones and stresses. ZoERFs have plenty of binding sites of ZoPP2Cs, suggesting a potential synergistic contribution between ZoERFs and ZoPP2Cs towards regulating growth/development and adverse conditions. The protein-protein interaction network displayed that five ZoPP2Cs (9/23/26/49/92) proteins have robust interaction relationship and potential function as hub proteins. Furthermore, the RNA-Seq and qRT-PCR analyses have shown that ZoPP2Cs exhibit various expression patterns during ginger maturation and responses to environmental stresses such as chilling, drought, flooding, salt, and Fusarium solani. Notably, exogenous application of melatonin led to notable up-regulation of ZoPP2Cs (17/59/11/72/43) under chilling stress. CONCLUSIONS: Taken together, our investigation provides significant insights of the ginger PP2C gene family and establishes the groundwork for its functional validation and genetic engineering applications.

MePP2C24, a cassava (Manihot esculenta) gene encoding protein phosphatase 2C, negatively regulates drought stress and abscisic acid responses in transgenic Arabidopsis thaliana.

Abscisic acid (ABA) signaling plays a crucial role in plant development and response to abiotic/biotic stress. However, the function and regulation of protein phosphatase 2C (PP2C), a key component of abscisic acid signaling, under abiotic stress are still unknown in cassava, a drought-tolerant crop. In this study, a cassava PP2C gene (MePP2C24) was cloned and characterized. The MePP2C24 transcripts increased in response to mannitol, NaCl, and ABA. Overexpression of MePP2C24 in Arabidopsis resulted in increased sensitivity to drought stress and decreased sensitivity to exogenous ABA. This was demonstrated by transgenic lines having higher levels of malondialdehyde (MDA), ion leakage (IL), and reactive oxygen species (ROS), lower activities of catalase (CAT) and peroxidase (POD), and lower proline content than wild type (WT) under drought stress. Moreover, MePP2C24 overexpression caused decrease in expression of drought-responsive genes related to ABA signaling pathway. In addition, MePP2C24 was localized in the cell nucleus and showed self-activation. Furthermore, many MePYLs (MePYL1, MePYL4, MePYL7-9, and MePYL11-13) could interact with MePP2C24 in the presence of ABA, and MePYL1 interacted with MePP2C24 in both the presence and absence of ABA. Additionally, MebZIP11 interacted with the promoter of MePP2C24 and exerted a suppressive effect. Taken together, our results suggest that MePP2C24 acts as a negative regulator of drought tolerance and ABA response.

Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.).

The protein phosphatase 2C (PP2C), a key regulator of the ABA signaling pathway, plays important roles in plant growth and development, hormone signaling, and abiotic stress response. Although the PP2C gene family has been identified in many species, systematic analysis was still relatively lacking in ramie (Boehmeria nivea L.). In the present study, we identified 63 BnPP2C genes from the ramie genome, using bioinformatics analysis, and classified them into 12 subfamilies, and this classification was consistently supported by their gene structures and conserved motifs. In addition, we observed that the functional differentiation of the BnPP2C family of genes was restricted and that fragment replication played a major role in the amplification of the BnPP2C gene family. The promoter cis-regulatory elements of BnPP2C genes were mainly involved in light response regulation, phytohormone synthesis, transport and signaling, environmental stress response and plant growth and development regulation. We identified BnPP2C genes with tissue specificity, using ramie transcriptome data from different tissues, in rhizome leaves and bast fibers. The qRT-PCR results showed that the BnPP2C1, BnPP2C26 and BnPP2C27 genes had a strong response to drought, high salt and ABA, and there were a large number of stress-responsive elements in the promoter region of BnPP2C1 and BnPP2C26. The results suggested that BnPP2C1 and BnPP2C26 could be used as the candidate genes for drought and salt tolerance in ramie. These results provide a reference for further studies on the function of the PP2C gene and advance the development of the mechanism of ramie stress response, with a view to providing candidate genes for the molecular breeding of ramie for drought and salt tolerance.

Genome-Wide Identification and Characterization of the PP2C Family from Zea mays and Its Role in Long-Distance Signaling.

The protein phosphatase 2C (PP2C) constitutes a large gene family that plays crucial roles in regulating stress responses and plant development. A recent study has shown the involvement of an AtPP2C family member in long-distance nitrogen signaling in Arabidopsis. However, it remains unclear whether maize adopts a similar mechanism. In this study, we conducted a genome-wide survey and expression analysis of the PP2C family in maize. We identified 103 ZmPP2C genes distributed across 10 chromosomes, which were further classified into 11 subgroups based on an evolutionary tree. Notably, cis-acting element analysis revealed the presence of abundant hormone and stress-related, as well as nitrogen-related, cis-elements in the promoter regions of ZmPP2Cs. Expression analysis demonstrated the distinct expression patterns of nine genes under two nitrogen treatments. Notably, the expression of ZmPP2C54 and ZmPP2C85 in the roots was found to be regulated by long-distance signals from the shoots. These findings provide valuable insights into understanding the roles of ZmPP2Cs in long-distance nitrogen signaling in maize.

Genome-wide identification of PYL/PYR-PP2C (A)-SnRK2 genes in Eutrema and their co-expression analysis in response to ABA and abiotic stresses.

ABA signaling core components PYR/PYL, group A PP2C and SnRK2 play important roles in various environmental stress responses of plants. This study identified 14 PYR/PYL, 9 PP2C (A), and 10 SnRK2 genes from halophytic Eutrema. Phylogenetic analysis showed 4 EsPYR/PYL, 4 EsPP2C (A) and 3 EsSnRK2 subfamilies characterized, which was supported by their gene structures and protein motifs. Large-scale segmental duplication event was demonstrated to be a major contributor to expansion of the EsPYL-PP2C (A)-SnRK2 gene families. Synteny relationship analysis revealed more orthologous PYL-PP2C (A)-SnRK2 gene pairs located in collinear blocks between Eutrema and Brassica than that between Eutrema and Arabidopsis. RNA-seq and qRT-PCR revealed EsABI1, EsABI2 and EsHAL2 showed a significantly up-regulated expression in leaves and roots in response to ABA, NaCl or cold stress. Three markedly co-expression modules of ABA/R-brown, NaCl/L-lightsteelblue1 and Cold/R-lightgreen were uncovered to contain EsPYL-PP2C (A)-SnRK2 genes by WGCNA analysis. GO and KEGG analysis indicated that the genes of ABA/R-brown module containing EsHAB1, EsHAI2 and EsSnRK2.6 were enriched in proteasome pathway. Further, EsHAI2-OE transgenic Arabidopsis lines showed significantly enhanced seeds germination and seedlings growth. This work provides a new insight for elucidating potential molecular functions of PYL-PP2C (A)-SnRK2 responding to ABA and abiotic stresses.

PP2C phosphatases Ptc1 and Ptc2 dephosphorylate PGK1 to regulate autophagy and aflatoxin synthesis in the pathogenic fungus Aspergillus flavus.

IMPORTANCE: Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Silencing ZmPP2C-A10 with a foxtail mosaic virus (FoMV) derived vector benefits maize growth and development following water limitation.

Global climate change is causing more frequent and severe droughts, which can have negative impacts on plant growth and crop productivity. Under drought conditions, plants produce the hormone ABA (abscisic acid), which regulates adaptive responses, such as stomatal closure and root elongation. Plant viruses have been used in the lab to convey new traits to plants and could also be used to increase production of ABA or to enhance downstream plant drought resistance responses. In this study, foxtail mosaic virus (FoMV) was used to silence ZmPP2C-A10, a negative regulator of ABA signalling, in maize (Zea mays L.). Both silenced and control plants were exposed to an 8-day drought treatment, followed by a 30-day period of rewatering, after which indicators of drought resistance were measured. After drought treatment, we observed a nearly twofold increase in expression of a stress-mitigation gene, ZmRAB17, reduced chlorophyll fluorescence changes (indicator of stress), and increased plant biomass and development in the ZmPP2C-A10-silenced maize compared to controls. These results demonstrate that the FoMV system can be used to silence endogenous expression of ZmPP2C-A10 and increase maize tolerance to drought. This could offer a useful tool to improve crop traits and reduce yield loss during the growing season.

Genome-Wide Identification of the PP2C Gene Family and Analyses with Their Expression Profiling in Response to Cold Stress in Wild Sugarcane.

Type 2C protein phosphatases (PP2Cs) represent a major group of protein phosphatases in plants, some of which have already been confirmed to play important roles in diverse plant processes. In this study, analyses of the phylogenetics, gene structure, protein domain, chromosome localization, and collinearity, as well as an identification of the expression profile, protein-protein interaction, and subcellular location, were carried out on the PP2C family in wild sugarcane (Saccharum spontaneum). The results showed that 145 PP2C proteins were classified into 13 clades. Phylogenetic analysis suggested that SsPP2Cs are evolutionarily closer to those of sorghum, and the number of SsPP2Cs is the highest. There were 124 pairs of SsPP2C genes expanding via segmental duplications. Half of the SsPP2C proteins were predicted to be localized in the chloroplast (73), with the next most common predicted localizations being in the cytoplasm (37) and nucleus (17). Analysis of the promoter revealed that SsPP2Cs might be photosensitive, responsive to abiotic stresses, and hormone-stimulated. A total of 27 SsPP2Cs showed cold-stress-induced expressions, and SsPP2C27 (Sspon.01G0007840-2D) and SsPP2C64 (Sspon.03G0002800-3D) were the potential hubs involved in ABA signal transduction. Our study presents a comprehensive analysis of the SsPP2C gene family, which can play a vital role in the further study of phosphatases in wild sugarcane. The results suggest that the PP2C family is evolutionarily conserved, and that it functions in various developmental processes in wild sugarcane.

Gypenosides Synergistically Reduce the Extracellular Matrix of Hepatic Stellate Cells and Ameliorate Hepatic Fibrosis in Mice.

Liver fibrosis resulting from chronic liver damage is becoming one of the major threats to health worldwide. Active saponin constituents isolated from Gynostemma pentaphyllum were found to possess a protective effect in liver diseases. Here, we obtained a naturally abundant gypenoside, XLVI, and evaluated its liver protection activity in both animal and cellular models. The results showed that it ameliorated acute and chronic liver injuries and lightened the process of fibrogenesis in vivo. XLVI can inhibit TGF-ß-induced activation of hepatic stellate cells and ECM deposition in vitro. The underlying mechanism study verified that it upregulated the protein expression of protein phosphatase 2C alpha and strengthened the vitality of the phosphatase together with a PP2Cα agonist gypenoside NPLC0393. These results shed new light on the molecular mechanisms and the potential therapeutic function of the traditional herb Gynostemma pentaphyllum in the treatment of liver fibrosis.

Arabidopsis Protein Phosphatase PIA1 Impairs Plant Drought Tolerance by Serving as a Common Negative Regulator in ABA Signaling Pathway.

Reversible phosphorylation of proteins is a ubiquitous regulatory mechanism in vivo that can respond to external changes, and plays an extremely important role in cell signal transduction. Protein phosphatase 2C is the largest protein phosphatase family in higher plants. Recently, it has been found that some clade A members can negatively regulate ABA signaling pathways. However, the functions of several subgroups of Arabidopsis PP2C other than clade A have not been reported, and whether other members of the PP2C family also participate in the regulation of ABA signaling pathways remains to be studied. In this study, based on the previous screening and identification work of PP2C involved in the ABA pathway, the clade F member PIA1 encoding a gene of the PP2C family, which was down-regulated after ABA treatment during the screening, was selected as the target. Overexpression of PIA1 significantly down-regulated the expression of ABA marker gene RD29A in Arabidopsis protoplasts, and ABA-responsive elements have been found in the cis-regulatory elements of PIA1 by promoter analysis. When compared to Col-0, transgenic plants overexpressing PIA1 were less sensitive to ABA, whereas pia1 showed the opposite trait in seed germination, root growth, and stomatal opening experiments. Under drought stress, SOD, POD, CAT, and APX activities of PIA1 overexpression lines were lower than Col-0 and pia1, while the content of H2O2 was higher, leading to its lowest survival rate in test plants, which were consistent with the significant inhibition of the expression of ABA-dependent stress-responsive genes RD29B, ABI5, ABF3, and ABF4 in the PIA1 transgenic background after ABA treatment. Using yeast two-hybrid and luciferase complementation assays, PIA1 was found to interact with multiple ABA key signaling elements, including 2 RCARs and 6 SnRK2s. Our results indicate that PIA1 may reduce plant drought tolerance by functioning as a common negative regulator involved in ABA signaling pathway.

Genome-wide identification of core components of ABA signaling and transcriptome analysis reveals gene circuits involved in castor bean (Ricinus communis L.) response to drought.

Castor bean (Ricinus communis L.) can withstand long periods of water deficit and high temperatures, and therefore has been recognized as a drought-resistant plant species, allowing the study of gene networks involved in drought response and tolerance. The identification of genes networks related to drought response in this plant may yield important information in the characterization of molecular mechanisms correlating changes in the gene expression with the physiological adaptation processes. In this context, gene families related to abscisic acid (ABA) signaling play a crucial role in developmental and environmental adaptation processes of plants to drought stress. However, the families that function as the core components of ABA signaling, as well as genes networks related to drought response, are not well understood in castor bean. In this study 7 RcPYL, 63 RcPP2C, and 6 RcSnRK2 genes were identified in castor bean genome, which was further supported by chromosomal distribution, gene structure, evolutionary relationships, and conserved motif analyses. The castor bean general expression profile was investigated by RNAseq in root and leaf tissues in response to drought stress. These analyses allowed the identification of genes differentially expressed, including genes from the ABA signaling core, genes related to photosynthesis, cell wall, energy transduction, antioxidant response, and transcription factors. These analyses provide new insights into the core components of ABA signaling in castor bean, allow the identification of several molecular responses associated with the high physiological adaptation of castor bean to drought stress, and contribute to the identification of candidate genes for genetic improvement.

DIW1 encoding a clade I PP2C phosphatase negatively regulates drought tolerance by de-phosphorylating TaSnRK1.1 in wheat.

Drought seriously impacts wheat production (Triticum aestivum L.), while the exploitation and utilization of genes for drought tolerance are insufficient. Leaf wilting is a direct reflection of drought tolerance in plants. Clade A PP2Cs are abscisic acid (ABA) co-receptors playing vital roles in the ABA signaling pathway, regulating drought response. However, the roles of other clade PP2Cs in drought tolerance, especially in wheat, remain largely unknown. Here, we identified a gain-of-function drought-induced wilting 1 (DIW1) gene from the wheat Aikang 58 mutant library by map-based cloning, which encodes a clade I protein phosphatase 2C (TaPP2C158) with enhanced protein phosphatase activity. Phenotypic analysis of overexpression and CRISPR/Cas9 mutant lines demonstrated that DIW1/TaPP2C158 is a negative regulator responsible for drought resistance. We found that TaPP2C158 directly interacts with TaSnRK1.1 and de-phosphorylates it, thus inactivating the TaSnRK1.1-TaAREB3 pathway. TaPP2C158 protein phosphatase activity is negatively correlated with ABA signaling. Association analysis suggested that C-terminal variation of TaPP2C158 changing protein phosphatase activity is highly correlated with the canopy temperature, and seedling survival rate under drought stress. Our data suggest that the favorable allele with lower phosphatase activity of TaPP2C158 has been positively selected in Chinese breeding history. This work benefits us in understanding the molecular mechanism of wheat drought tolerance, and provides elite genetic resources and molecular markers for improving wheat drought tolerance.

Genome-wide characterization of the PP2C gene family in peanut (Arachis hypogaea L.) and the identification of candidate genes involved in salinity-stress response.

Plant protein phosphatase 2C (PP2C) play important roles in response to salt stress by influencing metabolic processes, hormone levels, growth factors, etc. Members of the PP2C family have been identified in many plant species. However, they are rarely reported in peanut. In this study, 178 PP2C genes were identified in peanut, which were unevenly distributed across the 20 chromosomes, with segmental duplication in 78 gene pairs. AhPP2Cs could be divided into 10 clades (A-J) by phylogenetic analysis. AhPP2Cs had experienced segmental duplications and strong purifying selection pressure. 22 miRNAs from 14 different families were identified, targeting 57 AhPP2C genes. Gene structures and motifs analysis exhibited PP2Cs in subclades AI and AII had high structural and functional similarities. Phosphorylation sites of AhPP2C45/59/134/150/35/121 were predicted in motifs 2 and 4, which located within the catalytic site at the C-terminus. We discovered multiple MYB binding factors and ABA response elements in the promoter regions of the six genes (AhPP2C45/59/134/150/35/121) by cis-elements analysis. GO and KEGG enrichment analysis confirmed AhPP2C-A genes in protein binding, signal transduction, protein modification process response to abiotic stimulus through environmental information processing. Based on RNA-Seq data of 22 peanut tissues, clade A AhPP2Cs showed a varying degree of tissue specificity, of which, AhPP2C35 and AhPP2C121 specifically expressed in seeds, while AhPP2C45/59/134/150 expressed in leaves and roots. qRT-PCR indicated that AhPP2C45 and AhPP2C134 displayed significantly up-regulated expression in response to salt stress. These results indicated that AhPP2C45 and AhPP2C134 could be candidate PP2Cs conferring salt tolerance. These results provide further insights into the peanut PP2C gene family and indicate PP2Cs potentially involved in the response to salt stress, which can now be further investigated in peanut breeding efforts to obtain cultivars with improved salt tolerance.

PP2Cα aggravates neuronal insulin resistance leading to AD-like phenotype in vitro.

Neuronal insulin resistance is a major risk for development of Alzheimer's Disease (AD). Studies already reported few kinases participating in neuronal insulin signaling connected with progression of AD pathogenesis, yet complete information is missing. α isoform of Protein Phosphatase-2C (PP2C) is a Ser/Thr phosphatase, only known in 3T3-L1 adipocytes as a positive regulator of insulin signaling. However, many aspects of its function in neuronal insulin signaling and insulin resistance are unidentified. Recently, we reported that PP2Cα positively regulates neuronal glucose uptake possibly by a mechanism of dephosphorylation of IRS-1 at Ser522 and by inactivating AMPK, exacerbating hyperinsulinemia mediated neuronal insulin resistance. Since PP2Cα affected neuronal insulin signaling and AD is connected to neuronal insulin resistance, in the present study, we studied the role of PP2Cα in regulating activities of both isoforms of GSK3α and GSK3ß (one of the leading kinases for AD progression). The results led us to test the role of PP2Cα on AD hallmarks. Silencing of PP2Cα caused hyperphosphorylation of a potential kinase Tau, leading to NFT formation and increased Aß deposition. Our study thereby demonstrates escalation of hyperinsulinemia mediated neuronal insulin resistance leading to AD-like pathogenesis by PP2Cα in vitro and hints a novel molecule, PP2Cα, linking AD pathogenesis.

Pepper SnRK2.6-activated MEKK protein CaMEKK23 is directly and indirectly modulated by clade A PP2Cs in response to drought stress.

The phytohormone abscisic acid (ABA) is important for the plant growth and development, in which it plays a key role in the responses to drought stress. Among the core components of ABA signaling, SnRK2s interact with a range of proteins, including Raf-like MAP3Ks. In this study, we isolated the pepper MEKK subfamily member CaMEKK23 that interacts with CaSnRK2.6. CaMEKK23 has kinase activity and is specifically trans-phosphorylated by CaSnRK2.6. Compared with control plants, CaMEKK23-silenced pepper were found to be sensitive to drought stress and insensitive to ABA, whereas overexpression of CaMEKK23 in both pepper and Arabidopsis plants induced the opposite phenotypes. These altered phenotypes were established to be dependent on the kinase activity of CaMEKK23, which was also shown to interact with CaPP2Cs, functioning upstream of CaSnRK2.6. In addition to inhibiting the kinase activity of CaMEKK23, these CaPP2Cs were found to have inhibitory effects on CaSnRK2.6. Using CaMEKK23-, CaAITP1/CaMEKK23-, CaSnRK2.6-, and CaAITP1/CaSnRK2.6-silenced pepper, we revealed that CaMEKK23 and CaSnRK2.6 function downstream of CaAITP1. Collectively, our findings indicate that CaMEKK23 plays a positive regulatory role in the ABA-mediated drought stress responses in pepper plants, and that its phosphorylation status is modulated by CaSnRK2.6 and CaPP2Cs, functioning as core components of ABA signaling.

Genome-Wide Identification, Expression and Interaction Analyses of PP2C Family Genes in Chenopodium quinoa.

Plant protein phosphatase 2Cs (PP2Cs) function as inhibitors in protein kinase cascades involved in various processes and are crucial participants in both plant development and signaling pathways activated by abiotic stress. In this study, a genome-wide study was conducted on the CqPP2C gene family. A total of putative 117 CqPP2C genes were identified. Comprehensive analyses of physicochemical properties, chromosome localization and subcellular localization were conducted. According to phylogenetic analysis, CqPP2Cs were divided into 13 subfamilies. CqPP2Cs in the same subfamily had similar gene structures, and conserved motifs and all the CqPP2C proteins had the type 2C phosphatase domains. The expansion of CqPP2Cs through gene duplication was primarily driven by segmental duplication, and all duplicated CqPP2Cs underwent evolutionary changes guided by purifying selection. The expression of CqPP2Cs in various tissues under different abiotic stresses was analyzed using RNA-seq data. The findings indicated that CqPP2C genes played a role in regulating both the developmental processes and stress responses of quinoa. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis of six CqPP2C genes in subfamily A revealed that they were up-regulated or down-regulated under salt and drought treatments. Furthermore, the results of yeast two-hybrid assays revealed that subfamily A CqPP2Cs interacted not only with subclass III CqSnRK2s but also with subclass II CqSnRK2s. Subfamily A CqPP2Cs could interact with CqSnRK2s in different combinations and intensities in a variety of biological processes and biological threats. Overall, our results will be useful for understanding the functions of CqPP2C in regulating ABA signals and responding to abiotic stress.