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Background: Fucoidan, a polysaccharide derived from brown seaweed, has multiple biological properties, including antioxidant, antitumor, anticoagulant, and anti-inflammatory effects. However, there are few reports on their neuroprotective effects. Objective: This study is about the neuroprotective effects of fucoidan against Aβ42-induced neuronal apoptosis in human neuroblastoma SH-SY5Y cells. Methods: The protective effects of fucoidan against cell death by Aβ42 were measured with FDA/PI and Hoechst 33342 staining and immunoblotting about apoptosis-related proteins. Results: In this study, fucoidan activated autophagy by regulating the expression of autophagy markers, significantly upregulating the Bcl-2/Bax ratio, and downregulating cleaved caspase-3/caspase-3. Moreover, fucoidan improved Aβ42-induced neuronal apoptosis by activating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and mitogen-activated protein kinase cascade in SH-SY5Y cells. Additionally, Hoechst 33342 staining showed that fucoidan diminished the level of the apoptotic nuclei in Aβ42-induced SH-SY5Ycells. Conclusion: This study proposes fucoidan as a potential therapeutic candidate for AD treatment. [ABSTRACT FROM AUTHOR]

Serine kinases, also identified as mitogen-activated protein kinases (MAPKs), are involved in the transmission of cellular signals that carry out different cellular processes, i.e., mitosis, gene expression, cell proliferation, survival, and death. Moreover, it also leads to various other physiological processes in plants including metabolism, differentiation, stress response, and apoptosis. The MAPK family of proteins is responsible for initiating four distinct pathways through their components: ERK1/2 (extracellular signal-regulated kinase), c-Jun N-terminal kinases, p38 MAPK, and the stress activated protein kinases including ERK5 as a member of this family. Arabidopsis is generally used as a standard plant for determining the nomenclature of plant MAPK, and MAPK kinases. MAPK cascades contain certain protein kinase tiers which are activated by phosphorylating each other upon exposure to extracellular stimuli including, ERK1/2, ERK5/BMK1, p38, and JNK/SAPK signals. MAPK cascades also regulate multiple physiological functions by triggering immune system responses in multiple activities of various species of plants including, anther development, embryogenesis, zygote elongation, organ abscission, stomata pattering, and root development. MAPK has been reported in plant physiology regulation in responses to various biotic and abiotic stresses like cold, salt, metal, mechanical stress, ozone, extreme temperature, UV radiation, and insect, and pathogen infections. This process is mediated by hormones and phytochemicals including, ethylene and jasmonic acids which help to protect the plant from these environmental effects. Numerous studies on MAPK cascades have been performed in the past few decades and several MAPK cascades in plants have been identified, but certain details of MAPK regulation by upstream signaling components remain unknown. In the near future, MAPK cascades activated by external signals and unidentified MAPKs will be important research topics for most researchers. [ABSTRACT FROM AUTHOR]

Panicle exsertion is one of the crucial agronomic traits in rice (Oryza sativa). Shortening of panicle exsertion often leads to panicle enclosure and severely reduces seed production. Gibberellin (GA) plays important roles in regulating panicle exsertion. However, the underlying mechanism and the relative regulatory network remain elusive. Here, we characterized the oswrky78 mutant showing severe panicle enclosure, and found that the defect of oswrky78 is caused by decreased bioactive GA contents. Biochemical analysis demonstrates that OsWRKY78 can directly activate GA biosynthesis and indirectly suppress GA metabolism. Moreover, we found OsWRKY78 can interact with and be phosphorylated by mitogen‐activated protein kinase (MAPK) kinase OsMAPK6, and this phosphorylation can enhance OsWRKY78 stability and is necessary for its biological function. Taken together, these results not only reveal the critical function of OsWRKY78, but also reveal its mechanism via mediating crosstalk between MAPK and the GA signaling pathway in regulating panicle exsertion. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) cascades act as crucial signaling modules that regulate plant growth and development, response to biotic/abiotic stresses, and plant immunity. MAP3Ks can be activated through MAP4K phosphorylation in non-plant systems, but this has not been reported in plants to date. Here, we identified a total of 234 putative TaMAPK family members in wheat (Triticum aestivum L.). They included 48 MAPKs, 17 MAP2Ks, 144 MAP3Ks, and 25 MAP4Ks. We conducted systematic analyses of the evolution, domain conservation, interaction networks, and expression profiles of these TaMAPK–TaMAP4K (representing TaMAPK, TaMAP2K, TaMAP3K, and TaMAP4K) kinase family members. The 234 TaMAPK–TaMAP4Ks are distributed on 21 chromosomes and one unknown linkage group (Un). Notably, 25 of these TaMAP4K family members possessed the conserved motifs of MAP4K genes, including glycine-rich motif, invariant lysine (K) motif, HRD motif, DFG motif, and signature motif. TaMAPK3 and 6, and TaMAP4K10/24 were shown to be strongly expressed not only throughout the growth and development stages but also in response to drought or heat stress. The bioinformatics analyses and qRT-PCR results suggested that wheat may activate the MAP4K10–MEKK7–MAP2K11–MAPK6 pathway to increase drought resistance in wheat, and the MAP4K10–MAP3K8–MAP2K1/11-MAPK3 pathway may be involved in plant growth. In general, our work identified members of the MAPK–MAP4K cascade in wheat and profiled their potential roles during their response to abiotic stresses and plant growth based on their expression pattern. The characterized cascades might be good candidates for future crop improvement and molecular breeding. [ABSTRACT FROM AUTHOR]

Abstract Plants have evolved a complex and elaborate signaling network to respond appropriately to the pathogen invasion by regulating expression of defensive genes through certain transcription factors. The APETALA2/ethylene response factor (AP2/ERF) family members have been determined as key regulators in growth, development, and stress responses in plants. Moreover, a growing body of evidence has demonstrated the critical roles of AP2/ERFs in plant disease resistance. In this review, we describe recent advances for the function of AP2/ERFs in defense responses against microbial pathogens. We summarize that AP2/ERFs are involved in plant disease resistance by acting downstream of mitogen activated protein kinase (MAPK) cascades, and regulating expression of genes associated with hormonal signaling pathways, biosynthesis of secondary metabolites, and formation of physical barriers in an MAPK-dependent or -independent manner. The present review provides a multidimensional perspective on the functions of AP2/ERFs in plant disease resistance, which will facilitate the understanding and future investigation on the roles of AP2/ERFs in plant immunity.

Plants have evolved a complex and elaborate signaling network to respond appropriately to the pathogen invasion by regulating expression of defensive genes through certain transcription factors. The APETALA2/ethylene response factor (AP2/ERF) family members have been determined as key regulators in growth, development, and stress responses in plants. Moreover, a growing body of evidence has demonstrated the critical roles of AP2/ERFs in plant disease resistance. In this review, we describe recent advances for the function of AP2/ERFs in defense responses against microbial pathogens. We summarize that AP2/ERFs are involved in plant disease resistance by acting downstream of mitogen activated protein kinase (MAPK) cascades, and regulating expression of genes associated with hormonal signaling pathways, biosynthesis of secondary metabolites, and formation of physical barriers in an MAPK-dependent or -independent manner. The present review provides a multidimensional perspective on the functions of AP2/ERFs in plant disease resistance, which will facilitate the understanding and future investigation on the roles of AP2/ERFs in plant immunity. [ABSTRACT FROM AUTHOR]

Summary: Surface‐localized pattern recognition receptors perceive pathogen‐associated molecular patterns (PAMPs) to activate pattern‐triggered immunity (PTI). Activation of mitogen‐activated protein kinases (MAPKs) represents a major PTI response.Here, we report that Arabidopsis thaliana PIF3 negatively regulates plant defense gene expression and resistance to Pseudomonas syringae DC3000. PAMPs trigger phosphorylation of PIF3. Further study reveals that PIF3 interacts with and is phosphorylated by MPK3/6.By mass spectrometry and site‐directed mutagenesis, we identified the corresponding phosphorylation sites which fit for SP motif. We further show that a phospho‐mimicking PIF3 variant (PIF36D/pifq) conferred increased susceptibility to P. syringae DC3000 and caused lower levels of defense gene expression in plants.Together, this study reveals that PIF3 is phosphorylated by MPK3/6 and phosphorylation of the SP motif residues is required for its negative regulation on plant immunity. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) cascades act as crucial signaling modules that regulate plant growth and development, response to biotic/abiotic stresses, and plant immunity. MAP3Ks can be activated through MAP4K phosphorylation in non-plant systems, but this has not been reported in plants to date. Here, we identified a total of 234 putative TaMAPK family members in wheat (Triticum aestivum L.). They included 48 MAPKs, 17 MAP2Ks, 144 MAP3Ks, and 25 MAP4Ks. We conducted systematic analyses of the evolution, domain conservation, interaction networks, and expression profiles of these TaMAPK–TaMAP4K (representing TaMAPK, TaMAP2K, TaMAP3K, and TaMAP4K) kinase family members. The 234 TaMAPK–TaMAP4Ks are distributed on 21 chromosomes and one unknown linkage group (Un). Notably, 25 of these TaMAP4K family members possessed the conserved motifs of MAP4K genes, including glycine-rich motif, invariant lysine (K) motif, HRD motif, DFG motif, and signature motif. TaMAPK3 and 6, and TaMAP4K10/24 were shown to be strongly expressed not only throughout the growth and development stages but also in response to drought or heat stress. The bioinformatics analyses and qRT-PCR results suggested that wheat may activate the MAP4K10–MEKK7–MAP2K11–MAPK6 pathway to increase drought resistance in wheat, and the MAP4K10–MAP3K8–MAP2K1/11-MAPK3 pathway may be involved in plant growth. In general, our work identified members of the MAPK–MAP4K cascade in wheat and profiled their potential roles during their response to abiotic stresses and plant growth based on their expression pattern. The characterized cascades might be good candidates for future crop improvement and molecular breeding.

As a multifunctional phytohormone, melatonin (Mel) plays pivotal roles in plant responses to multiple stresses. However, its mechanism of action remains elusive. In the present study, we evaluated the role of NO and Ca2+ signaling in Mel enhanced cold tolerance in winter turnip rape. The results showed that the NO content and concentration of intracellular free Ca2+ ([Ca2+] cyt) increased by 35.42% and 30.87%, respectively, in the leaves of rape seedlings exposed to cold stress. Compared with those of the seedlings in cold stress alone, the NO content and concentration of [Ca2+] cyt in rape seedlings pretreated with Mel increased further. In addition, the Mel-mediated improvement of cold tolerance was inhibited by L-NAME (a NO synthase inhibitor), tungstate (a nitrate reductase inhibitor), LaCl 3 (a Ca2+ channel blocker), and EGTA (a Ca2+ chelator), and this finding was mainly reflected in the increase in ROS content and the decrease in osmoregulatory capacity, photosynthetic efficiency and antioxidant enzyme activities, and expression levels of antioxidant enzyme genes. These findings suggest that NO and Ca2+ are necessary for Mel to improve cold tolerance and function synergistically downstream of Mel. Notably, the co-treatment of Mel with L-NAME, tungstate, LaCl 3 , or EGTA also inhibited the Mel-induced expression of MAPK3/6 under cold stress. In conclusion, NO and Ca2+ are involved in the enhancement of cold tolerance induced by Mel through activating the MAPK cascades in rape seedlings, and a crosstalk may exist between NO and Ca2+ signaling. [Display omitted] • Melatonin (Mel) caused the accumulation of NO and [Ca2+] cyt. • Inhibition of NO and Ca2+ signaling inverted the positive effect of melatonin. • NO, Ca2+ and MAPK cascades participated in melatonin-induced cold tolerance. [ABSTRACT FROM AUTHOR]

Leaf blight, caused by Botrytis elliptica (Berk.) Cooke, is a devastating disease that limits the production of Lilium in China and in other countries worldwide. Numerous studies have indicated that plants have evolved sophisticated and effective signal transduction and defense-related pathways in response to pathogen invasion. Recently, particular attention has been given to the action(s) of melatonin in plants in response to biotic stress, and the role of melatonin in plant–pathogen interactions has also been discussed. In this study, RNA-seq was applied to analyze the transcriptomic changes in Lilium leaves that were pre-treated and post-treated with melatonin after B. elliptica infection for 0, 12, 24, 36, and 72 h and then compare those changes with those of the control. Treatment with exogenous melatonin and infection with B. elliptica caused differential expression of a large number of genes in Lilium leaves. KEGG pathway analysis showed that, after melatonin treatment, the defense-related DEGs were mainly enriched in plant–pathogen interactions, plant hormone signal transduction, MAPK signaling pathways, phenylpropanoid biosynthesis, and phenylalanine metabolism. RT–qPCR was used to verify the expression changes of 12 DEGs, the results of which were consistent with the RNA-seq analysis results. The expression of DEGs related to the MAPK pathway were significantly different between the MB group and the HB group, suggesting that, via the MAPK signaling cascade, melatonin may play a role in the disease resistance of Lilium to B. elliptica. This study provides a new perspective and information for molecular-based breeding of Lilium disease resistance. [ABSTRACT FROM AUTHOR]

Leaf blight, caused by Botrytis elliptica (Berk.) Cooke, is a devastating disease that limits the production of Lilium in China and in other countries worldwide. Numerous studies have indicated that plants have evolved sophisticated and effective signal transduction and defense-related pathways in response to pathogen invasion. Recently, particular attention has been given to the action(s) of melatonin in plants in response to biotic stress, and the role of melatonin in plant–pathogen interactions has also been discussed. In this study, RNA-seq was applied to analyze the transcriptomic changes in Lilium leaves that were pre-treated and post-treated with melatonin after B. elliptica infection for 0, 12, 24, 36, and 72 h and then compare those changes with those of the control. Treatment with exogenous melatonin and infection with B. elliptica caused differential expression of a large number of genes in Lilium leaves. KEGG pathway analysis showed that, after melatonin treatment, the defense-related DEGs were mainly enriched in plant–pathogen interactions, plant hormone signal transduction, MAPK signaling pathways, phenylpropanoid biosynthesis, and phenylalanine metabolism. RT–qPCR was used to verify the expression changes of 12 DEGs, the results of which were consistent with the RNA-seq analysis results. The expression of DEGs related to the MAPK pathway were significantly different between the MB group and the HB group, suggesting that, via the MAPK signaling cascade, melatonin may play a role in the disease resistance of Lilium to B. elliptica. This study provides a new perspective and information for molecular-based breeding of Lilium disease resistance.

Abstract Background The contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites. Results In this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes’ behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones. Conclusion The data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Abstract Background Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. Results The ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 μM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP. Conclusions The EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense response. However, the molecular mechanisms underlying MAPK cascades are still very elusive, largely because of our poor understanding of how they relay the signals. The MAPK cascade is composed of MAPK, MAPKK, and MAPKKK. They transfer signals through the phosphorylation of MAPKKK, MAPKK, and MAPK in turn. MAPKs are organized into a complex network for efficient transmission of specific stimuli. This review summarizes the research progress in recent years on the classification and functions of MAPK cascades under various conditions in plants, especially the research status and general methods available for identifying MAPK substrates, and provides suggestions for future research directions.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling transduction module that transduces extracellular stimuli into intracellular responses in plants. Early studies of plant MAPKs focused on their functions in model plants. Based on the results of whole-genome sequencing, many MAPKs have been identified in horticultural plants, such as tomato and apple. Recent studies revealed that the MAPK cascade also plays crucial roles in the biotic and abiotic stress responses of horticultural plants. In this review, we summarize the composition and classification of MAPK cascades in horticultural plants and recent research on this cascade in responses to abiotic stresses (such as drought, extreme temperature and high salinity) and biotic stresses (such as pathogen infection). In addition, we discuss the most advanced research themes related to plant MAPK cascades, thus facilitating research on MAPK cascade functions in horticultural plants.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling transduction module that transduces extracellular stimuli into intracellular responses in plants. Early studies of plant MAPKs focused on their functions in model plants. Based on the results of whole-genome sequencing, many MAPKs have been identified in horticultural plants, such as tomato and apple. Recent studies revealed that the MAPK cascade also plays crucial roles in the biotic and abiotic stress responses of horticultural plants. In this review, we summarize the composition and classification of MAPK cascades in horticultural plants and recent research on this cascade in responses to abiotic stresses (such as drought, extreme temperature and high salinity) and biotic stresses (such as pathogen infection). In addition, we discuss the most advanced research themes related to plant MAPK cascades, thus facilitating research on MAPK cascade functions in horticultural plants. [ABSTRACT FROM AUTHOR]

Background: Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. Results: The ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 μM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP. Conclusions: The EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation. [ABSTRACT FROM AUTHOR]

The role of leptin has been documented in several studies, including activated threonine phosphorylation of extracellular signal-regulated kinase (ERK1/2) in the reproduction of rodents and humans. Our previous studies have demonstrated that mitogen-activated protein kinase (MAPK) cascades ERK, P38, and c-Jun N-terminal kinase (JNK) are involved in the spermatogenesis and acrosome reaction of Eriocheir sinensis. Therefore, the aim of this study was to investigate the expression of leptin and its receptor (LepR), and the effect of leptin on MAPK cascades during calcium ionophore A23187-induced spermatozoa acrosome reaction in crabs. Successful western blotting revealed a 16 kDa band for leptin, and 120 kDa and 90 kDa bands for the obese receptor (LepR), respectively, in the tested male reproductive tissues. Both leptin and LepR were localized at the pro-acrosomal vesicle and apical cap (AC) of spermatids, suggesting their role in the subsequent acrosome reaction. Moreover, acrosome reaction can be enhanced by leptin, and this effect decreased due to the anti-LepR antibody. Afterwards, we investigated the effects of leptin on MAPK cascades. The results showed that leptin mainly activated the phosphorylation of ERK, P38 and JNK proteins in the apical cap during the acrosome reaction in crab spermatozoa. This study addresses the role of leptin on spermatozoa, and suggests that leptin may induce molecular changes associated with spermatozoa during acrosome reaction. [ABSTRACT FROM AUTHOR]

Excessive inflammatory response and oxidative stress are the leading causes of periodontal disease (PD). Previously, we reported that Lactobacillus paracasei subsp. paracasei NTU 101-fermented skim milk ethanol extract (NTU101FSMEE) may be useful for developing anti-periodontitis products. In this study, the bioactive ingredient in NTU101FSMEE was isolated and identified as a mixture of tyrosine and lactic acid in 3:1 ratio (3T1L). Treatment of RAW 264.7 and HGF-1 cells with 3T1L significantly reduced the levels of various cytokines participating in the inflammatory response of PD. 3T1L inhibited osteoclastogenesis by decreasing the tartrate-resistant acid phosphatase-related index and suppressed bone resorption by regulating matrix metalloproteinase‐9 and tissue inhibitor of metalloproteinase‐1 levels. Furthermore, 3T1L modulated the intracellular oxidative stress and expression of mitogen-activated protein kinases and the nuclear factor-κB signaling pathway observed in response to lipopolysaccharide. Our findings suggest the potential of 3T1L derived from NTU101FSMEE for anti-periodontitis application.

This study investigates the effects of γ-Aminobutyric acid (GABA) and mitogen-activated protein kinase (MAPK) inhibitor (PD98059) in disease resistance in red pitaya fruit. Results demonstrated that GABA treatment suppressed the growth of Gilbertella persicaria ; increased H 2 O 2 content and O 2 •- production rate; enhanced reduced glutathione and ascorbic acid contents, as well as increased the activities and transcripts of catalase (CAT), NADPH oxidase (NOX), peroxidise (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and up-regulated HcMYB111 / 24 expressions. Furthermore, GABA promoted the expressions of HcMAPKKK1 - HcMAPKK2 / 4 - HcMAPK1 / 7 cascades. However, PD98059 +GABA treatment decreased the disease resistance against G. persicaria and reduced H 2 O 2 content; inhibited the activities and gene expressions of NOX, CAT, SOD, POD, GR and APX, and down-regulated the expressions of HcMAPK1 / 7. These findings suggest that GABA induces disease resistance by regulating ROS metabolism and MAPK cascades in red pitaya fruit. • GABA treatment reduced postharvest disease in red pitaya fruit. • PD98059 +GABA treatment increased disease incidence in red pitaya fruit. • GABA treatment induced ROS homeostasis to enhance disease resistance. • GABA mediated the activation of MAPK cascade to enhance antioxidant systems. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the effect of six chlorogenic acid (CGA) isomers known to be present in coffee and other plant foods on modulating the inflammatory response induced by pro-inflammatory cytokines in the Caco-2 human intestinal epithelial cell line. Compared to caffeoylquinic acids (CQA), dicaffeoylquinic acids (DiCQA) had significantly stronger (p < 0.05) capacities to reduce phosphorylation of one of mitogen-activated protein kinases (MAPK) cascades, namely p38. Compared to the control, CQA isomers treatment resulted in around 50% reduction in an interleukin-8 (IL-8) secretion, whereas DiCQA, at the same concentration, resulted in a 90% reduction in IL-8 secretion, compared to the control cells. CGA isomer treatment also showed a significant effect (p < 0.05) on the up-regulation of NFκB subunit p65 nuclear translocation by more than 1.5 times, compared to the control. We concluded that CGA isomers exert anti-inflammatory activity in a mixture of interferon gamma (IFNγ) and phorbol myristate acetate (PMA)-challenged Caco-2 cells, by decreasing the phosphorylation of p38 cascade and up-regulating NFκB signaling.

In 2007, we reported that a phytopathogen effector directly inhibits a MAP kinase cascade. In the decade since, many more effectors have been found to inhibit MAP kinase cascades, providing not only a mechanistic understanding of pathogenesis and immunity in plants, but also the identification of previously unknown enzymes. [ABSTRACT FROM AUTHOR]

The fungal kingdom displays an extraordinary diversity of lifestyles, developmental processes, and ecological niches. The MAPK (mitogen-activated protein kinase) cascade consists of interlinked MAPKKK, MAPKK, and MAPK, and collectively such cascades play pivotal roles in cellular regulation in fungi. However, the mechanism by which evolutionarily conserved MAPK cascades regulate diverseoutput responses in fungi remains unknown. Here we identified the full complement of MAPK cascade components from 231 fungal species encompassing 9 fungal phyla. Using the largest data set to date, we found that MAPK family members could have two ancestors, while MAPKK and MAPKKK family members could have only one ancestor. The current MAPK, MAPKK, and MAPKKK subfamilies resulted from duplications and subsequent subfunctionalization during the emergence of the fungal kingdom. However, the gene structure diversification and gene expansion and loss have resulted in significant diversity in fungal MAPK cascades, correlating with the evolution of fungal species and lifestyles. In particular, a distinct evolutionary trajectory of MAPK cascades was identified in single-celled fungi in the Saccharomycetes. All MAPK, MAPKK, and MAPKKK subfamilies expanded in the Saccharomycetes; genes encoding MAPK cascade components have a similar exon-intron structure in this class that differs from those in other fungi. [ABSTRACT FROM AUTHOR]

Fungal necrotrophic pathogens cause widespread crop losses and infect a variety of plants. The perception of these pathogens or their associated signals by specific receptors in plants triggers the mitogen-activated protein kinase (MAPK) cascades and activates hormone (jasmonates and ethylene)-dependent and hormone-independent signaling, which facilitates the mounting of a defense response against the invading necrotrophs. This response involves the activation of specific transcription factors that result in the production of antifungal proteins (plant defensins) or accumulation of defensive secondary metabolites (phytoalexins). The perception and communication mechanisms triggered by pathogen-associated molecular patterns and the hormones are coordinated by the MAPK signaling cascades which integrate various aspects of the multi-layered plant defense response. This review focuses on compiling distinct and overlapping roles played by various components of the plant signaling machinery in recognizing and mounting a regulated defense response against necrotrophic fungal pathogens. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf and 6 to ZIK subfamily respectively. Conserved protein motif, exon-intron organization and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and functional characterization of MAPKKK family in Brachypodium, which will facilitate the functional analysis of BdMAPKKK genes, and also will facilitate better understanding the MAPK signal pathway in Brachypodium and beyond.

Exposure of plants to toxic concentrations of metals leads to disruption of the cellular redox status followed by an accumulation of reactive oxygen species (ROS). ROS, like hydrogen peroxide, can act as signaling molecules in the cell and induce signaling via mitogen-activated protein kinase (MAPK) cascades. MAPK cascades are evolutionary conserved signal transduction modules, able to convert extracellular signals to appropriate cellular responses. In this review, our current understanding about MAPK signaling in plant metal stress is discussed. However, this knowledge is scarce compared to research into the role of MAPK signaling in the case of other abiotic and biotic stresses. ROS production is a common response induced by different stresses and undiscovered analogies may exist with metal stress. Therefore, further attention is given to MAPK signaling in other biotic and abiotic stresses and its interplay with other signaling pathways to create a framework in which the involvement of MAPK signaling in metal stress may be studied.

Background: Signal transduction is the process through which cells communicate with the external environment, interpret stimuli and respond to them. This mechanism is controlled by signaling cascades, which play the role of intracellular transmitter, being able to transmit biochemical information between cell membrane and nucleus. In theory as well as in practice, it has been shown that a perturbation can propagate upstream (and not only downstream) a cascade, by a mechanism known as retroactivity. This study aims to compare the conditions on biochemical parameters which favor one or the other direction of signaling in such a cascade. Results: From a mathematical point of view, we show that the steady states of a cascade of arbitrary length n are described by an iterative map of second order, meaning that the cascade tiers are actually coupled three-by-three. We study the influence of the biochemical parameters in the control of the direction of transmission - upstream and/or downstream - along a signaling cascade. A numerical and statistical approach, based on the random scan of parameters describing a 3-tier signaling cascade, provides complementary findings to the analytical study. In particular, computing the likelihood of parameters with respect to various signaling regimes, we identify conditions on biochemical parameters which enhance a specific direction of propagation corresponding to forward or retro-signaling regimes. A compact graphical representation is designed to relay the gist of these conditions. Conclusions: The values of biochemical parameters such as kinetic rates, Michaelis-Menten constants, total concentrations of kinases and of phosphatases, determine the propensity of a cascade to favor or impede downstream or upstream signal transmission. We found that generally there is an opposition between parameter sets favoring forward and retro-signaling regimes. Therefore, on one hand our study supports the idea that in most cases, retroactive effects can be neglected when a cascade which is efficient in forward signaling, is perturbed by an external ligand inhibiting the activation at some tier of the cascade. This result is relevant for therapeutic methodologies based on kinase inhibition. On the other hand, our study highlights a less-known part of the parameter space where, although the forward signaling is inefficient, the cascade can interestingly act as a retro-signaling device. [ABSTRACT FROM AUTHOR]

Jasmonates (JAs) [Jasmonic acid (JA) and methyl jasmonates (MeJAs)] are known to take part in various physiological processes. Exogenous application of JAs so far tested on different plants under abiotic stresses particularly salinity, drought, and temperature (low/high) conditions have proved effective in improving plant stress tolerance. However, its extent of effectiveness entirely depends on the type of plant species tested or its concentration. The effects of introgression or silencing of different JA- and Me-JA-related genes have been summarized in this review, which have shown a substantial role in improving crop yield and quality in different plants under stress or non-stress conditions. Regulation of JAs synthesis is impaired in stressed as well as unstressed plant cells/tissues, which is believed to be associated with a variety of metabolic events including signal transduction. Although, mitogen activated protein kinases (MAPKs) are important components of JA signaling and biosynthesis pathways, nitric oxide, ROS, calcium, ABA, ethylene, and salicylic acid are also important mediators of plant growth and development during JA signal transduction and synthesis. The exploration of other signaling molecules can be beneficial to examine the details of underlying molecular mechanisms of JA signal transduction. Much work is to be done in near future to find the proper answers of the questions like action of JA related metabolites, and identification of universal JA receptors etc. Complete signaling pathways involving MAPKs, CDPK, TGA, SIPK, WIPK, and WRKY transcription factors are yet to be investigated to understand the complete mechanism of action of JAs. [ABSTRACT FROM AUTHOR]

Phenolic acids are the major bioactive metabolites produced in Salvia miltiorrhiza , a traditional Chinese medicine called Danshen. Many phytohormone elicitor treatments induce phenolic acid biosynthesis, even though the underlying mechanism remains obscure. Expression pattern analysis showed that SmMAPK3 was highly expressed in leaves, and SmMAPK3 was significantly induced by salicylic acid (SA) and methyl jasmonate (JA). Bioinformatics analysis revealed that SmMAPK3 belongs to group A and contains a TEY motif in the activation loop together with three conserved regions (P-loop, C-loop and CD-domain). A previous study speculated that SmMAPK3 is likely a positive regulator in the biosynthesis of phenolic acids in S. miltiorrhiza. In this study, overexpression of SmMAPK3 increased phenolic acid biosynthetic gene expression and enhanced the accumulation of phenolic acids in S. miltiorrhiza plantlets. Yeast two-hybrid (Y2H) analysis and firefly luciferase complementation imaging (LCI) assays revealed that SmMAPKK2/4/5/7-SmMAPK3-SmJAZs form a cascade that regulates the accumulation of phenolic acids. In summary, this work deepens our understanding of the posttranscriptional regulatory mechanisms of phenolic acid biosynthesis and sheds new light on metabolic engineering in S. miltiorrhiza. To response the treatment of SA and MeJA, SmMAPK3, the substrate of SmMAPKK2/4/5/7, reduces the stability of transcription repressor SmJAZs/IAAs via phosphorylation and consequently enhances the expression of secondary metabolite genes in S. miltiorrhiza. The SmMAPKK2/4/5/7-SmMAPK3-SmJAZs cascade plays a crucial regulatory role in phenolic acids biosynthesis. [Display omitted] • SmMAPK3 responses to phytohormone SA and MeJA. • Overexpression of SmMAPK3 upregulates phenolic acid biosynthesis. • SmMAPK3 interacts with upstream kinase SmMAPKK2/4/5/7 and downstream transcription repressor JAZs. • The SmMAPKK2/4/5/7-SmMAPK3-SmJAZs cascade regulates secondary metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules in animals, plants and yeast. MAPK cascades are complicated networks and play vital roles in signal transduction pathways involved in biotic and abiotic stresses. In this study, a maize MAPKK gene, ZmMKK1, was characterized. Quantitative real time PCR (qRT-PCR) analysis demonstrated that ZmMKK1 transcripts were induced by diverse stresses and ABA signal molecule in maize root. Further study showed that the ZmMKK1-overexpressing Arabidopsis enhanced the tolerance to salt and drought stresses. However, seed germination, post-germination growth and stomatal aperture analysis demonstrated that ZmMKK1 overexpression was sensitive to ABA in transgenic Arabidopsis. Molecular genetic analysis revealed that the overexpression of ZmMKK1 in Arabidopsis enhanced the expression of ROS scavenging enzyme- and ABA-related genes, such as POD, CAT, RAB18 and RD29A under salt and drought conditions. In addition, heterologous overexpression of ZmMKK1 in yeast (Saccharomyces cerevisiae) improved the tolerance to salt and drought stresses. These results suggested that ZmMKK1 might act as an ABA- and ROS-dependent protein kinase in positive modulation of salt and drought tolerance. Most importantly, ZmMKK1 interacted with ZmMEKK1 as evidenced by yeast two-hybrid assay, redeeming a deficiency of MAPK interaction partners in maize. [Copyright &y& Elsevier]

Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense response. However, the molecular mechanisms underlying MAPK cascades are still very elusive, largely because of our poor understanding of how they relay the signals. The MAPK cascade is composed of MAPK, MAPKK, and MAPKKK. They transfer signals through the phosphorylation of MAPKKK, MAPKK, and MAPK in turn. MAPKs are organized into a complex network for efficient transmission of specific stimuli. This review summarizes the research progress in recent years on the classification and functions of MAPK cascades under various conditions in plants, especially the research status and general methods available for identifying MAPK substrates, and provides suggestions for future research directions. [ABSTRACT FROM AUTHOR]

Exposure of plants to toxic concentrations of metals leads to disruption of the cellular redox status followed by an accumulation of reactive oxygen species (ROS). ROS, like hydrogen peroxide, can act as signaling molecules in the cell and induce signaling via mitogen-activated protein kinase (MAPK) cascades. MAPK cascades are evolutionary conserved signal transduction modules, able to convert extracellular signals to appropriate cellular responses. In this review, our current understanding about MAPK signaling in plant metal stress is discussed. However, this knowledge is scarce compared to research into the role of MAPK signaling in the case of other abiotic and biotic stresses. ROS production is a common response induced by different stresses and undiscovered analogies may exist with metal stress. Therefore, further attention is given to MAPK signaling in other biotic and abiotic stresses and its interplay with other signaling pathways to create a framework in which the involvement of MAPK signaling in metal stress may be studied. [ABSTRACT FROM AUTHOR]

Abstract: The ERK cascade is a central signaling pathway that regulates a large number of intracellular processes including proliferation, differentiation, development and also survival or apoptosis. The induction of so many distinct and even opposing cellular processes raises the question as to how the signaling specificity of the cascade is regulated. In the past few years, subcellular localization of components of the ERK cascade was shown to play an important role in specificity determination. Here we describe the dynamic subcellular localization of Raf kinases, MEKs, and particularly ERKs, which translocate into the nucleus during many cellular processes to induce transcription. We also describe in details the recent identification of a novel nuclear translocation mechanism for ERKs, which is based on a nuclear translocation sequence (NTS) within their kinase insert domain (KID). Phosphorylation of this domain, mainly upon stimulation, allows ERKs to interact with the nuclear importing protein – importin7, which mediates the penetration of the interacting ERKs into the nucleus via nuclear pores. Interestingly, the NTS is not specific to ERKs, and seems to be a general signal for regulating nuclear accumulation of various proteins, including MEKs, upon their stimulation. Better understanding of this mechanism may clarify the role of the massive nuclear translocation of many regulatory proteins shortly after their stimulation. [Copyright &y& Elsevier]

In humans and animal models, the kidneys and cardiovascular systems are negatively affected by BPA from the environment. It is considered that BPA have some potential estrogen-like and non-hormone-like properties. In this study, RNA-sequencing and its-related bioinformatics was used as the basic strategy to clarify the characteristic mechanisms of kidney–heart axis remodeling and dysfunction in diabetic male rats under BPA exposure. We found that continuous BPA exposure in diabetic rats aggravated renal impairment, and caused hemodynamic disorders and dysfunctions. There were 655 and 125 differentially expressed genes in the kidney and heart, respectively. For the kidneys, functional annotation and enrichment, and gene set enrichment analyses identified bile acid secretion related to lipid synthesis and transport, and MAPK cascade pathways. For the heart, these bioinformatics analyses clearly pointed to MAPKs pathways. A total of 12 genes and another total of 6 genes were identified from the kidney tissue and heart tissue, respectively. Western blotting showed that exposure to BPA activated MAPK cascades in both organs. In this study, the exacerbated remodeling of diabetic kidney–heart axis under BPA exposure and diabetes might occur through hemodynamics, metabolism disorders, and the immune-inflammatory response, as well as continuous estrogen-like stimulation, with focus on the MAPK cascades. [Display omitted] • Environmental BPA exposure accelerated damage to the structures of both organs, aggravated renal impairment, and caused hemodynamic disorders in the status of diabetes mellitus. • The dual effects of chronic BPA exposure and diabetes mellitus were determined to exacerbate kidney–heart axis remodeling characterized by destruction and dysfunction. • The alterations of the axis accelerated by environmental BPA might occur through hemodynamics, metabolism, and the immune-inflammatory response, with focus on the MAPK cascades, and not completely depend on the estrogenic effect of BPA. This study to understand how environmental disrupting chemicals interact with pathologies associated with diabetes is an important topic, and the use of the metabolic disease model to examine the effects of BPA exposure and diabetes on alternations of kidney-heart axis is innovative. [ABSTRACT FROM AUTHOR]

A hallmark of protein kinase/phosphatase cascades, including mitogen-activated protein kinase (MAPK) pathways, is the spatial separation of their components within cells. The top-level kinase, MAP3K, is phosphorylated at the cell membrane, and cytoplasmic kinases at sequential downstream levels (MAP2K and MAPK) spread the signal to distant targets. Given measured protein diffusivity and phosphatase activities, signal propagation by diffusion would result in a steep decline of MAP2K activity and low bisphosphorylated MAPK (ppMAPK) levels near the nucleus, especially in large cells, such as oocytes. Here, we show that bistability in a two-site MAPK (de)phosphorylation cycle generates a novel type of phosphoprotein wave that propagates from the surface deep into the cell interior. Positive feedback from ppMAPK to cytoplasmic MAP2K enhances the propagation span of the ppMAPK wave, making it possible to convey phosphorylation signals over exceedingly long distances. The finding of phosphorylation waves traveling with constant amplitude and high velocity may solve a long-standing enigma of survival signaling in developing neurons. [ABSTRACT FROM AUTHOR]

Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on three subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. However, the ERK family is broader than the ERK1 and ERK2 proteins that have been the subject of most studies in this area. Here we overview the work on ERKs 3 to 8, emphasising where possible their biological activities as well as distinctive biochemical properties. It is clear from these studies that these additional ERKs show similarities to ERK1 and ERK2, but with some interesting differences that challenge the paradigm of the archetypical ERK1/2 MAPK pathway. [Copyright &y& Elsevier]

The notions of asymptotic amplitude for signals, and Cauchy gain for input/output systems, and an associated small-gain principle, are introduced. These concepts allow the consideration of systems with multiple, and possibly feedback-dependent, steady states. A Lyapunov-like characterization allows the computation of gains for state-space systems, and the formulation of sufficient conditions insuring the lack of oscillations and chaotic behaviors in a wide variety of cascades and feedback loops. An application in biology (MAPK signaling) is worked out in detail. [Copyright &y& Elsevier]

• An anti-periodontitis mixture of tyrosine and lactic acid (3T1L) was identified. • 3T1L abolished bone resorption through RANKL-induced osteoclastogenesis. • 3T1L regulated MAPK signal phosphorylation and NF-κB to improve periodontitis. • NTU101FSMEE has potential to be used as a functional food supplement. Excessive inflammatory response and oxidative stress are the leading causes of periodontal disease (PD). Previously, we reported that Lactobacillus paracasei subsp. paracasei NTU 101-fermented skim milk ethanol extract (NTU101FSMEE) may be useful for developing anti-periodontitis products. In this study, the bioactive ingredient in NTU101FSMEE was isolated and identified as a mixture of tyrosine and lactic acid in 3:1 ratio (3T1L). Treatment of RAW 264.7 and HGF-1 cells with 3T1L significantly reduced the levels of various cytokines participating in the inflammatory response of PD. 3T1L inhibited osteoclastogenesis by decreasing the tartrate-resistant acid phosphatase-related index and suppressed bone resorption by regulating matrix metalloproteinase‐9 and tissue inhibitor of metalloproteinase‐1 levels. Furthermore, 3T1L modulated the intracellular oxidative stress and expression of mitogen-activated protein kinases and the nuclear factor-κB signaling pathway observed in response to lipopolysaccharide. Our findings suggest the potential of 3T1L derived from NTU101FSMEE for anti-periodontitis application. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the effect of six chlorogenic acid (CGA) isomers known to be present in coffee and other plant foods on modulating the inflammatory response induced by pro-inflammatory cytokines in the Caco-2 human intestinal epithelial cell line. Compared to caffeoylquinic acids (CQA), dicaffeoylquinic acids (DiCQA) had significantly stronger (p < 0.05) capacities to reduce phosphorylation of one of mitogen-activated protein kinases (MAPK) cascades, namely p38. Compared to the control, CQA isomers treatment resulted in around 50% reduction in an interleukin-8 (IL-8) secretion, whereas DiCQA, at the same concentration, resulted in a 90% reduction in IL-8 secretion, compared to the control cells. CGA isomer treatment also showed a significant effect (p < 0.05) on the up-regulation of NFκB subunit p65 nuclear translocation by more than 1.5 times, compared to the control. We concluded that CGA isomers exert anti-inflammatory activity in a mixture of interferon gamma (IFNγ) and phorbol myristate acetate (PMA)-challenged Caco-2 cells, by decreasing the phosphorylation of p38 cascade and up-regulating NFκB signaling. [ABSTRACT FROM AUTHOR]

Summary Mitogen-activated protein kinase cascades are important signaling modules that convert environmental stimuli into cellular responses. We show that MPK3, MPK4, and MPK6 are rapidly activated after cold treatment. The mpk3 and mpk6 mutants display increased expression of CBF genes and enhanced freezing tolerance, whereas constitutive activation of the MKK4/5-MPK3/6 cascade in plants causes reduced expression of CBF genes and hypersensitivity to freezing, suggesting that the MKK4/5-MPK3/6 cascade negatively regulates the cold response. MPK3 and MPK6 can phosphorylate ICE1, a basic-helix-loop-helix transcription factor that regulates the expression of CBF genes, and the phosphorylation promotes the degradation of ICE1. Interestingly, the MEKK1-MKK2-MPK4 pathway constitutively suppresses MPK3 and MPK6 activities and has a positive role in the cold response. Furthermore, the MAPKKK YDA and two calcium/calmodulin-regulated receptor-like kinases, CRLK1 and CRLK2, negatively modulate the cold activation of MPK3/6. Our results uncover important roles of MAPK cascades in the regulation of plant cold response. [ABSTRACT FROM AUTHOR]

The fungal kingdom displays an extraordinary diversity of lifestyles, developmental processes, and ecological niches. The MAPK (mitogen-activated protein kinase) cascade consists of interlinked MAPKKK, MAPKK, and MAPK, and collectively such cascades play pivotal roles in cellular regulation in fungi. However, the mechanism by which evolutionarily conserved MAPK cascades regulate diverse output responses in fungi remains unknown. Here we identified the full complement of MAPK cascade components from 231 fungal species encompassing 9 fungal phyla. Using the largest data set to date, we found that MAPK family members could have two ancestors, while MAPKK and MAPKKK family members could have only one ancestor. The current MAPK, MAPKK, and MAPKKK subfamilies resulted from duplications and subsequent subfunctionalization during the emergence of the fungal kingdom. However, the gene structure diversification and gene expansion and loss have resulted in significant diversity in fungal MAPK cascades, correlating with the evolution of fungal species and lifestyles. In particular, a distinct evolutionary trajectory of MAPK cascades was identified in single-celled fungi in the Saccharomycetes. All MAPK, MAPKK, and MAPKKK subfamilies expanded in the Saccharomycetes; genes encoding MAPK cascade components have a similar exon-intron structure in this class that differs from those in other fungi., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Activation of MAPK (Mitogen-Activated Protein Kinase) cascades after MAMP (Microbe-Associated Molecular Pattern) perception through PRR (Pattern Recognition Receptor) is one of the first conserved responses when plants encounter microbial organisms. Phosphorylation of various cellular factors in the MAMP-PRR pathway by MAPK cascades is critical for broad-spectrum plant innate immunity. Measurement of MAPK activation and identification of MAPK phosphorylation targets in the MAMP-PRR signal transduction pathway are essential to understand how plants reprogram their cellular processes to cope with unfavorable microbial attack. Here, we describe detailed protocols of three assays measuring MAPK activity after MAMP perception: (1) immune-blotting analysis with anti-phospho ERK1/2 antibody; (2) in-gel kinase assay using a general substrate myelin basic protein (MBP); (3) an in vitro kinase assay to evaluate phosphorylation of MAPK substrate candidates during MAMP-PRR signaling based on a protoplast expression system.

Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium . Phylogenetic analysis of MAPKKK family from Arabidopsis , rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium , while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and functional characterization of MAPKKK family in Brachypodium , which will facilitate the functional analysis of BdMAPKKK genes, and also will facilitate better understanding the MAPK signal pathway in Brachypodium and beyond.