Stress-induced Mitogen-activated Protein Kinase Research Articles

Abstract B049: Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity

Abstract Epidemiologic studies have shown that obesity is a risk factor for more than a dozen cancers, including pancreatic ductal adenocarcinoma (PDAC). As PDAC is one of the leading causes of cancer-related death and obesity rates are rapidly increasing worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. To address this gap in knowledge, our lab combined a well-established genetic model for obesity (O: Lepob/ob) with a Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) that mimics human PDAC progression. Resultant KCO mice had a drastic increase in tumor development compared to their lean counterparts, whereas induced weight loss intercepted tumor progression, showing that the tumor-promoting effects of obesity can be reversed. Bulk RNA sequencing of tumors from these mice revealed aberrantly increased expression of multiple peptide hormones expressed in insulin-producing beta (b) cells of pancreatic islets, including cholecystokinin (CCK) and amylin (Iapp). Strikingly, transgenic expression of CCK and Iapp in b-cells was sufficient to promote Kras-driven pancreatic tumorigenesis in non-obese mice. Single-cell RNA sequencing of b cells showed a trajectory of changes in gene expression with increasing obesity from wildtype to high-fat diet-fed to Lepob/ob mice. Further in silico analysis combining archetypal (AANet) with pseudotime (TrajectoryNet) methods demonstrated that b cells misexpressing hormones (CCK and Iapp) arose from a specific subtype of preexisting b cells, specifically virgin b cells, a rare, immature, regenerative population. Preliminary in vivo lineage tracing analyses in MIP-CreERT;LSL-TdTomato;Lepob/ob mice support these findings. Finally, we investigated the transcriptional networks that lead to aberrant CCK and Iapp expression in b cells. By combining Grainger causality analysis with known gene-gene relationships in the Transcriptional Regulatory Relationship Unraveled by Sentence-based Text mining (TRRUST) v2 database, we discovered a stress-induced mitogen-activated protein kinase (MAPK8)-cJun pathway that promotes pro-tumorigenic hormone expression in b cells. These combined experimental and computational methods nominate novel b cell targets to subvert the development of PDAC, an exocrine tumor. Citation Format: Cathy C. Garcia, Aarthi Venkat, Alex Tong, Sherry Agabiti, Lauren Lawres, Rebecca Cardone, Richard Kibbey, Smita Krishnaswamy, Mandar D Muzumdar. Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B049.

Advanced microscopy resolves dynamic localization patterns of stress-induced mitogen-activated protein kinase (SIMK) during alfalfa root hair interactions with Ensifer meliloti.

Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

Injury-induced MAPK activation triggers body axis formation in Hydra by default Wnt signaling

Hydra's almost unlimited regenerative potential is based on Wnt signaling, but so far it is unknown how the injury stimulus is transmitted to discrete patterning fates in head and foot regenerates. We previously identified mitogen-activated protein kinases (MAPKs) among the earliest injury response molecules in Hydra head regeneration. Here, we show that three MAPKs-p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs)-are essential to initiate regeneration in Hydra, independent of the wound position. Their activation occurs in response to any injury and requires calcium and reactive oxygen species (ROS) signaling. Phosphorylated MAPKs hereby exhibit cross talk with mutual antagonism between the ERK pathway and stress-induced MAPKs, orchestrating a balance between cell survival and apoptosis. Importantly, Wnt3 and Wnt9/10c, which are induced by MAPK signaling, can partially rescue regeneration in tissues treated with MAPK inhibitors. Also, foot regenerates can be reverted to form head tissue by a pharmacological increase of β-catenin signaling or the application of recombinant Wnts. We propose a model in which a β-catenin-based stable gradient of head-forming capacity along the primary body axis, by differentially integrating an indiscriminate injury response, determines the fate of the regenerating tissue. Hereby, Wnt signaling acquires sustained activation in the head regenerate, while it is transient in the presumptive foot tissue. Given the high level of evolutionary conservation of MAPKs and Wnts, we assume that this mechanism is deeply embedded in our genome.

Photo-Protective and Anti-Inflammatory Effects of Antidesma thwaitesianum Müll. Arg. Fruit Extract against UVB-Induced Keratinocyte Cell Damage.

The main cause of most skin cancers is damage from UVB from sunlight, which penetrate the skin surface and induce inflammation. For this reason, this study aims to identify natural products with photo-protection properties and their mode of action by using the UVB-irradiated HaCaT keratinocyte model. Antidesma thwaitesianum fruit extracts at 25, 50, and 100 µg/mL recovered cell viability following UVB exposure in a dose-dependent manner. Cell survival was associated with the reduction in intracellular ROS and NO. In addition, we showed that the pre-treatment with the fruit extract lowered the phosphorylation level of two MAPK-signaling pathways: p38 MAPKs and JNKs. The resulting lower MAPK activation decreased their downstream pro-inflammatory cascade through COX-2 expression and subsequently reduced the PGE2 proinflammatory mediator level. The photoprotective effects of the fruit extract were correlated with the presence of polyphenolic compounds, including cyanidin, ferulic acid, caffeic acid, vanillic acid, and protocatechuic acid, which have been previously described as antioxidant and anti-inflammation. Together, we demonstrated that the pre-treatment with the fruit extract had photo-protection by inhibiting oxidative stress and subsequently lowered stress-induced MAPK responses. Therefore, this fresh fruit is worthy of investigation to be utilized as a skincare ingredient for preventing UVB-induced skin damage.

Dual control of MAPK activities by AP2C1 and MKP1 MAPK phosphatases regulates defence responses in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

Antioxidant Properties of Hydrogen Gas Attenuates Oxidative Stress in Airway Epithelial Cells.

Oxidative stress plays a crucial role in the development of airway diseases. Recently, hydrogen (H2) gas has been explored for its antioxidant properties. This study investigated the role of H2 gas in oxidative stress-induced alveolar and bronchial airway injury, where A549 and NCI-H292 cells were stimulated with hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) in vitro. Results show that time-dependent administration of 2% H2 gas recovered the cells from oxidative stress. Various indicators including reactive oxygen species (ROS), nitric oxide (NO), antioxidant enzymes (catalase, glutathione peroxidase), intracellular calcium, and mitogen-activated protein kinase (MAPK) signaling pathway were examined to analyze the redox profile. The viability of A549 and NCI-H292 cells and the activity of antioxidant enzymes were reduced following induction by H2O2 and LPS but were later recovered using H2 gas. Additionally, the levels of oxidative stress markers, including ROS and NO, were elevated upon induction but were attenuated after treatment with H2 gas. Furthermore, H2 gas suppressed oxidative stress-induced MAPK activation and maintained calcium homeostasis. This study suggests that H2 gas can rescue airway epithelial cells from H2O2 and LPS-induced oxidative stress and may be a potential intervention for airway diseases.

Pharmacological Inhibition of p38 MAPK Rejuvenates Bone Marrow Derived-Mesenchymal Stromal Cells and Boosts their Hematopoietic Stem Cell-Supportive Ability.

The therapeutic value of mesenchymal stromal cells (MSCs) for various regenerative medicine applications, including hematopoietic stem cell transplantations (HSCT), has been well-established. Owing to their small numbers in vivo, it becomes necessary to expand them in vitro, which leads to a gradual loss of their regenerative capacity. Stress-induced mitogen-activated protein kinase p38 (p38 MAPK) signaling has been shown to compromise the MSC functions. Therefore, we investigated whether pharmacological inhibition of p38 MAPK signaling rejuvenates the cultured MSCs and boosts their functionality. Indeed, we found that the ex vivo expanded MSCs show activated p38 MAPK signaling and exhibit increased oxidative stress. These MSCs show a decreased ability to secrete salutary niche factors, thereby compromising their ability to support hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation. We, therefore, attempted to rejuvenate the cultured MSCs by pharmacological inhibition of p38 MAPK - a strategy broadly known as "priming of MSCs". We demonstrate that priming of MSCs with a p-38 MAPK inhibitor, PD169316, boosts their niche-supportive functions via upregulation of various HSC-supportive transcription factors. These primed MSCs expand multipotent HSCs having superior homing and long-term reconstitution ability. These findings shed light on the significance of non-cell-autonomous mechanisms operative in the hematopoietic niche and point towards the possible use of pharmacological compounds for rejuvenation ofex vivocultured MSCs. Such approaches could improve the outcome of regenerative therapies involving in vitro cultured MSCs.

Overexpression of alfalfa SIMK promotes root hair growth, nodule clustering and shoot biomass production.

SummaryNitrogen‐fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen‐activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress‐induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well‐known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP‐tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP‐tagged SIMK led to increase of biomass in above‐ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.

Nephroprotective effect of wogonin against cadmium-induced nephrotoxicity via inhibition of oxidative stress-induced MAPK and NF-kB pathway in Sprague Dawley rats.

Oxidative stress (OS) is one of the responsible factors for causing renal diseases. For the treatment or prevention of the renal disease, antioxidants use could be a hopeful therapeutic mediation as they block the oxidative reaction along with inflammatory process. Wogonin (Wog) is a plant flavonoid, a pharmacologically active component of Scutellaria baicalensis Georgi (Huang Qui), which exhibits antioxidant activity. In this investigation, we explored the nephroprotective activity of Wog on cadmium (Cd)-induced nephron toxicity in rats. Administering (10 and 20 mg/kg) intraperitoneally diminished Cd-induced anomalies in kidney histology and creatinine and serum urea levels. Wog therapy reduced the Cd-influenced generation of inflammatory mediators, inclusive of tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta. Western blot analysis demonstrated that Wog abolished proinflammatory nuclear factor-kappa B (NF-κB) p65 stimulation, phosphorylation of p38 mitogen-activated protein kinases (MAPKs). In all, Wog demonstrated antioxidative and anti-inflammatory effects in Cd- intoxicated rats by obstructing OS and activation of NF-κB via restricting the stimulation of upstream kinases inclusive of MAPKs.

Adverse effects, expression of defense-related genes, and oxidative stress-induced MAPK pathway in the benzo[α]pyrene-exposed rotifer Brachionus rotundiformis

To examine the adverse effects of the benzo[α]pyrene (B[α]P), the monogonont rotifer Brachionus rotundiformis was exposed to various concentration of B[α]P (0 [control], 1, 10, and 100 μg/L) and measured life cycle parameters (e.g., mortality, fecundity [cumulated number of offspring], and lifespan), reactive oxygen species (ROS), antioxidant enzymatic activity of glutathione S-transferase (GST). In addition, defense-related transcripts (e.g., glutathione S-transferases [GSTs], ATP binding cassette [ABCs] transporters) and Western blot analysis of mitogen-activated protein kinase (MAPK) signaling pathway were investigated in B[α]P-exposed rotifer. In this study, the total intracellular ROS level and GST activity were significantly increased (P < 0.05), while fecundity and lifespan were also significantly (P < 0.05) reduced in a concentration dependent manner in B[α]P-exposed B. rotundiformis. In addition, transcriptional regulation of GSTs and ABC transporters were significantly upregulated and downregulated (P < 0.05), respectively, suggesting that B[α]P can induce oxidative stress leading to induction of antioxidant system and detoxification mechanism. In addition to detoxification-related genes, B[α]P-exposed B. rotundiformis showed the increased levels of the p-JNK and p-p38, suggesting that B[α]P can activate MAPK signaling pathway in B. rotundiformis.

Growth, Physiological, Biochemical, and Transcriptional Responses to Drought Stress in Seedlings of Medicago sativa L., Medicago arborea L. and Their Hybrid (Alborea)

Medicago sativa L. is a tetraploid perennial forage legume of great agronomical interest. The increasing need for its use under water-deficit conditions as well as low-input systems demands further improvement of its drought tolerance. On the other hand, Medicagoarborea L. is a perennial leguminous shrub, which is knownas a drought-tolerant species. In the present study, drought stress responses of the aforementioned medicago species, along with their hybrid, named Alborea, were comparatively assayed at the morphological, physiological, biochemical, and transcriptional levels. In particular, transcript abundance of representative genes that: (a) control ion transport, intracellular Na+/H+ antiporters(NHX1) and rare cold inducible2A (RCI2A); (b) have an osmotic function Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1); and (c) participate in signaling pathways and control cell growth and leaf function stress-induced mitogen-activated protein kinases kinases (SIMKK), Zinc Finger (ZFN), apetala2/ethylene-responsive element binding (AP2/EREB), basic leucine zipper (bzip) and Medicago sativa Helicase 1(MH1) were evaluated. Under well-watered conditions, the studied population of Alborea showed the highest stem elongation rate and photosynthetic rate that were dramatically reduced under drought conditions compared to M. sativa and M. arborea. Under drought conditions, the studied population of M. arborea showed less reduction of relative water content, all gas-exchange parameters, less lipid peroxidation, and more antioxidant capacity. Moreover, transcriptional analysis demonstrated that the population of M. arborea exhibited significantly higher transcript levels of drought-responsive genes in both leaves and roots under drought stress conditions. M. sativa has better antioxidant capacity than Alborea and had a higher induction of stress-related genes, thus it performs better than Alborea under drought conditions. Among the studied genes, it seems that AP2/EREB play a critical role in the response of the studied population to drought stress.

The Role of c-Jun N-Terminal Kinase (JNK) in Retinal Degeneration and Vision Loss.

c-Jun N-terminal kinase (JNK), a member of stress-induced mitogen-activated protein (MAP) kinase family, has been shown to modulate a variety of biological processes associated with neurodegenerative pathology of the retina. In particular, various retinal cell culture and animal models related to glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa indicate that JNK signaling may contribute to disease pathogenesis. This mini-review discusses the impact of JNK signaling in retinal disease, with a focus on retinal ganglion cells (RGCs), photoreceptor cells, retinal pigment epithelial (RPE) cells, and animal studies, with particular attention to modulation of JNK signaling as a potential therapeutic target for the treatment of retinal disease.

Genome-wide identification of the MAPK gene family in chickpea and expression analysis during development and stress response

Mitogen-activated protein kinases (MAPKs) are universal cell signaling components amongst all the eukaryotes and govern critical roles in growth, development and environment adaptation. However, very little information is available about the MAPK family in chickpea. Here, we provide a comprehensive functional analysis of the Mitogen-activated protein kinases (MAPK) family from Cicer arietinum including identification and nomenclature, chromosomal localization, phylogenetic relationships, and investigation of expression analysis in different tissues and in response to abiotic stresses. A genome wide study identified 19 MAPK genes in the chickpea genome, and phylogenetic analysis showed that MAPK family members could be classified into four distinct groups; genes within the same groups had almost similar exon–intron structures. All MAPK proteins in groups A, B and C had a Thr-Glu-Tyr (TEY) activation domain, whereas those in group D contained a Thr-Asp-Tyr (TDY) activation domain. We explored transcriptome data and performed Quantitative real-time (QPCR) analysis, which showed that all the MAPKs were expressed more in flowers and young pods and are rapidly regulated by abiotic stresses and ABA treatment. There was a strong correlation between promoter cis-elements and stress-induced MAPK gene expression. These results contribute to a comprehensive knowledge on MAPKs and their regulation and further assist in functional characterization of MAPK cascades in chickpea.

Overexpression of F-box only protein 31 predicts poor prognosis and deregulates p38α- and JNK-mediated apoptosis in esophageal squamous cell carcinoma.

F-box only protein 31 (FBXO31), a subunit of the Skp1-Cul1-F box ubiquitin ligase, plays a crucial role in DNA damage response and tumorigenesis. Yet its expression and function vary in different types of human cancer. The expression of FBXO31 in esophageal squamous cell carcinoma (ESCC) and its association with clinicopathological features is not well studied. The underlying mechanism by which deregulated FBXO31 contributes to ESCC tumorigenesis is largely unknown. By immunohistochemical analysis of a tissue microarray containing 85 cases of ESCC and matched adjacent noncancerous tissue and an additional 10 cases of ESCC tissue samples, we found that FBXO31 was overexpressed in ESCC, and that its expression was significantly correlated with histological grade (p = 0.04) and clinical stage (p = 0.022). Higher expression of FBXO31 was associated with poor prognosis in univariate (p = 0.013) and multivariate (p = 0.014) analyses. We found that FBXO31 functioned as an antiapoptotic molecule in ESCC cells exposed to different types of genotoxic stress. Knockdown of FBXO31 inhibited serum-starved cell viability and decreased tumorigenicity of ESCC cells. In addition, the antiapoptotic effects of FBXO31 were associated with deactivation of stress-induced MAPK p38α and JNK. Furthermore, in vitro and in vivo data showed that silencing of FBXO31-sensitized ESCC cells and tumors to cisplatin treatment. Taken together, in addition to revealing that FBXO31 is an independent prognostic marker for ESCC, our findings substantiate a novel regulatory role of FBXO31 in tumorigenesis and drug resistance of ESCC.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

The complex genetic and molecular basis of a model quantitative trait.

Quantitative traits are often influenced by many loci with small effects. Identifying most of these loci and resolving them to specific genes or genetic variants is challenging. Yet, achieving such a detailed understanding of quantitative traits is important, as it can improve our knowledge of the genetic and molecular basis of heritable phenotypic variation. In this study, we use a genetic mapping strategy that involves recurrent backcrossing with phenotypic selection to obtain new insights into an ecologically, industrially, and medically relevant quantitative trait-tolerance of oxidative stress, as measured based on resistance to hydrogen peroxide. We examine the genetic basis of hydrogen peroxide resistance in three related yeast crosses and detect 64 distinct genomic loci that likely influence the trait. By precisely resolving or cloning a number of these loci, we demonstrate that a broad spectrum of cellular processes contribute to hydrogen peroxide resistance, including DNA repair, scavenging of reactive oxygen species, stress-induced MAPK signaling, translation, and water transport. Consistent with the complex genetic and molecular basis of hydrogen peroxide resistance, we show two examples where multiple distinct causal genetic variants underlie what appears to be a single locus. Our results improve understanding of the genetic and molecular basis of a highly complex, model quantitative trait.

Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants.

The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.

Dominant Repression by Arabidopsis Transcription Factor MYB44 Causes Oxidative Damage and Hypersensitivity to Abiotic Stress

In any living species, stress adaptation is closely linked with major changes of the gene expression profile. As a substrate protein of the rapidly stress-induced mitogen-activated protein kinase MPK3, Arabidopsis transcription factor MYB44 likely acts at the front line of stress-induced re-programming. We recently characterized MYB44 as phosphorylation-dependent positive regulator of salt stress signaling. Molecular events downstream of MYB44 are largely unknown. Although MYB44 binds to the MBSII element in vitro, it has no discernible effect on MBSII-driven reporter gene expression in plant co-transfection assays. This may suggest limited abundance of a synergistic co-regulator. MYB44 carries a putative transcriptional repression (Ethylene responsive element binding factor-associated Amphiphilic Repression, EAR) motif. We employed a dominant repressor strategy to gain insights into MYB44-conferred stress resistance. Overexpression of a MYB44-REP fusion markedly compromised salt and drought stress tolerance—the opposite was seen in MYB44 overexpression lines. MYB44-mediated resistance likely results from induction of tolerance-enhancing, rather than from repression of tolerance-diminishing factors. Salt stress-induced accumulation of destructive reactive oxygen species is efficiently prevented in transgenic MYB44, but accelerated in MYB44-REP lines. Furthermore, heterologous overexpression of MYB44-REP caused tissue collapse in Nicotiana. A mechanistic model of MAPK-MYB-mediated enhancement in the antioxidative capacity and stress tolerance is proposed. Genetic engineering of MYB44 variants with higher trans-activating capacity may be a means to further raise stress resistance in crops.

Quantification of stress-induced mitogen-activated protein kinase expressional dynamic using reverse transcription quantitative real-time PCR.

Although it is generally accepted that signal transduction in plant mitogen-activated protein kinase signaling cascades is regulated via rapid posttranslational modifications, there are also several compelling examples of swift stress induced transcriptional activation of plant MAP kinase genes. A possible function of these fast and transient events is to compensate for protein losses caused by degradation of phosphorylated MAP kinases within stimulated pathways. Nevertheless, there is still need for additional evidence to precisely describe the regulatory role of plant MAP kinase transcriptional dynamics, especially in the context of whole stress stimulated pathways including also other signaling molecules and transcription factors. During the last two decades a reverse transcription quantitative real-time PCR became a golden choice for the accurate and fast quantification of the gene expression and gene expression dynamic. In here, we provide a robust, cost-effective SYBR Green-based RT-qPCR protocol that is suitable for the quantification of stress induced plant MAP kinase transcriptional dynamics in various plant species.