AtHMGB15 regulates tapetal apoptosis in pollen development and actin dynamics during pollen germination in arabidopsis.

KEY MESSAGE: ARID-HMG DNA binding protein, AtHMGB15, regulates pollen development and pollen germination in Arabidopsis. Previous studies have shown that ARID-HMG DNA binding protein, AtHMGB15 regulate pollen development and pollen germination in Arabidopsis. Here, we performed transcriptome and cytological studies to understand the role of AtHMGB15 in regulating pollen wall morphology and the pollen tube germination rate. Our result showed abnormal vacuolization in the tapetal cells during anther maturation and prolonged PCD in AtHMGB15 loss-of-function mutant. The tapetum has the ability to perform both secretory and biosynthetic activities critical for pollen maturation and pollen viability. Interestingly, expression of PCD executer genes CEP1, MC9 and RNS3 were significant down-regulation of in athmgb15-4. The growth of pollen tubes is regulated by the actin cytoskeleton dynamics. To address the defect in pollen tube growth of athmgb15, we monitored the actin network in growing pollen tubes of wildtype and athmgb15-4 using Rhodamine-phalloidin fluorescence. Our results indicate a highly fragmented actin distribution in athmgb15-4 pollen tubes with a lesser number of long actin fibers and significantly low f-actin concentration at the apex. q-RTPCR further indicates significant downy-regulation of actin regulatory proteins VLN2 and PRF4. Collectively, our results suggest that AtHMGB15 being a nuclear architectural protein orchestrates high-order chromatin organization to promote the transcription of genes responsible for pollen development and pollen germination.

Dynamicity of histone H3K27ac and H3K27me3 modifications regulate the cold-responsive gene expression in Oryza sativa L. ssp. indica.

Cultivated in tropical and subtropical regions, Oryza sativa L. ssp. indica is largely affected by cold-stress, especially at the seedling stage. The present model of the stress-responsive regulatory network in plants entails the role of genetic and epigenetic factors in stress-responsive gene expression. Despite extensive transcriptomic studies, the regulation of various epigenetic factors in plants cold-stress response is less explored. The present study addresses the effect of genome-wide changes of H3K27 modifications on gene expression in IR64 rice, during cold-stress. Our results suggest a positive correlation between the changes in H3K27 modifications and stress-responsive gene activation in indica rice. Cold-induced enrichment of H3K27 acetylation promotes nucleosomal rearrangement, thereby facilitating the accessibility of the transcriptional machinery at the stress-responsive loci for transcription activation. Although H3K27ac exhibits uniform distribution throughout the loci of enriched genes; occupancy of H3K27me3 is biased to intergenic regions. Integration of the ChIP-seq data with transcriptome indicated that upregulation of stress-responsive TFs, photosynthesis-TCA-related, water-deficit genes, redox and JA signalling components, was associated with differential changes of H3K27ac and H3K27me3 levels. Furthermore, cold-induced upregulation of histone acetyltransferases and downregulation of DNA methyltransferases was noted through the antagonistic switch of H3K27ac and H3K27me3. Moreover, motif analysis of H3K27ac and H3K27me3 enriched regions are associated with putative stress responsive transcription factors binding sites, GAGA element and histone H3K27demethylase. Collectively our analysis suggests that differential expression of various chromatin and DNA modifiers coupled with increased H3K27ac and depleted H3K27me3 increases DNA accessibility, thereby promoting transcription of the cold-responsive genes in indica rice.

Identification of genome-wide targets and DNA recognition sequence of the Arabidopsis HMG-box protein AtHMGB15 during cold stress response.

AtHMGB15 belongs to a group of ARID-HMG proteins which are plant specific. The presence of two known DNA binding domains: AT rich interacting domain (ARID) and High Mobility Group (HMG)-box, in one polypeptide, makes this protein intriguing. Although proteins containing individual HMG and ARID domains have been characterized, not much is known about the role of ARID-HMG proteins. Promoter analysis of AtHMGB15 showed the presence of various stress responsive cis regulatory elements along with MADS-box containing transcription factors. Our result shows that the expression of AtHMGB15 increased significantly upon application of cold stress. Using ChIP-chip approach, we have identified 6128 and 4689 significantly enriched loci having AtHMGB15 occupancy under control and cold stressed condition respectively. GO analysis shows genes belonging to abiotic stress response, cold response and root development were AtHMGB15 targets during cold stress. DNA binding and footprinting assays further identified A(A/C)--ATA---(A/T)(A/T) as AtHMGB15 binding motif. The enriched probe distribution in both control and cold condition shows a bias of AtHMGB15 binding towards the transcribed (gene body) region. Further, the expression of cold stress responsive genes decreased in athmgb15 knockout plants compared to wild-type. Taken together, binding enrichment of AtHMGB15 to the promoter and upstream to stress loci suggest an unexplored role of the protein in stress induced transcription regulation.

Understanding the early cold response mechanism in IR64 indica rice variety through comparative transcriptome analysis.

BACKGROUND: Cellular reprogramming in response to environmental stress involves alteration of gene expression, changes in the protein and metabolite profile for ensuring better stress management in plants. Similar to other plant species originating in tropical and sub-tropical areas, indica rice is highly sensitive to low temperature that adversely affects its growth and grain productivity. Substantial work has been done to understand cold induced changes in gene expression in rice plants. However, adequate information is not available for early gene expression, especially in indica variety. Therefore, a transcriptome profile was generated for cold shock treated seedlings of IR64 variety to identify early responsive genes. RESULTS: The functional annotation of early DEGs shows enrichment of genes involved in altered membrane rigidity and electrolytic leakage, the onset of calcium signaling, ROS generation and activation of stress responsive transcription factors in IR64. Gene regulatory network suggests that cold shock induced Ca2+ signaling activates DREB/CBF pathway and other groups of transcription factors such as MYB, NAC and ZFP; for activating various cold-responsive genes. The analysis also indicates that cold induced signaling proteins like RLKs, RLCKs, CDPKs and MAPKK and ROS signaling proteins. Further, several late-embryogenesis-abundant (LEA), dehydrins and low temperature-induced-genes were upregulated under early cold shock condition, indicating the onset of water-deficit conditions. Expression profiling in different high yielding cultivars shows high expression of cold-responsive genes in Heera and CB1 indica varieties. These varieties show low levels of cold induced ROS production, electrolytic leakage and high germination rate post-cold stress, compared to IR36 and IR64. Collectively, these results suggest that these varieties may have improved adaptability to cold stress. CONCLUSIONS: The results of this study provide insights about early responsive events in Oryza sativa l.ssp. indica cv IR64 in response to cold stress. Our data shows the onset of cold response is associated with upregulation of stress responsive TFs, hydrophilic proteins and signaling molecules, whereas, the genes coding for cellular biosynthetic enzymes, cell cycle control and growth-related TFs are downregulated. This study reports that the generation of ROS is integral to the early response to trigger the ROS mediated signaling events during later stages.

Analysis of DNA Methylation Profile in Plants by Chop-PCR.

Plants, when challenged with any unfavorable condition, such as biotic or abiotic stress, adapt to the stress via physiological or structural changes. DNA methylation, an important epigenetic factor, plays an integral role in determining chromatin dynamicity and in turn regulates the process of gene transcription in eukaryotes. DNA methylation resulting in 5-methylcytosine interferes with the transcription process by hindering accessibility of the transcriptional machinery. Transcriptionally active genes are predominantly hypomethylated, whereas repressed genes exhibit hypermethylation. It can thus be interpreted that the presence of methylation in the promoter and upstream regions of loci represses their transcription and vice versa. Chop-PCR is a targeted DNA methylation detection technique that uses partial digestion by methylation-sensitive restriction enzymes (MSREs) followed by PCR amplification. The presence of cytosine methylation at the cleavage sites of the MSREs protects the DNA against digestion and therefore can be amplified using PCR. Enzymatic cleavage occurs unhindered at unmethylated restriction sites and subsequent PCR amplification of the target sequence is not observed.

Change in Nucleosome Dynamics During Stress Responses in Plants.

The dynamic nature of chromatin is the basis for the regulation of various biological processes in eukaryotic organisms. Nucleosome, the basic unit of chromatin in eukaryotes, undergo various reversible posttranslational modifications (PTM) in response to both external and internal cues. This PTM is recognized by different reader molecules, which facilitates the recruitment of various chromatin remodeling proteins that modulate the chromatin structure. In plants, the promoters of active genes are associated with higher lysine acetylation of histones H3 and H4, and these modifications are recognized by Bromo-domain (BRM) containing chromatin remodelers. This leads to the remodeling of the nucleosome at promoter regions, thereby increasing accessibility of the transcription machinery. It also plays a role in transcriptional repression when enriched in repressed genes. Lysine methylation recruits methyl-binding domain-containing proteins such as LIKE HETEROCHROMATIN PROTEIN1 (LHP1), which facilitates a more condensed chromatin structure that further inhibits access by the transcriptional machinery. In this article, protocols to study the regulation of chromatin conformations and nucleosome dynamics in plants in response to different stress signals are provided.

Rice Trithorax factor ULTRAPETALA 1 (OsULT1) specifically binds to "GAGAG" sequence motif present in Polycomb response elements.

Co-ordinated interplay between Polycomb group (PcG) and Trithorax group (TrxG) of proteins regulate chromatin state and maintain the transcription "off" and "on" state of a gene in higher eukaryotes. Targeting PcG complex to a specific locus is mediated by DNA sequences known as Polycomb response elements. Interestingly, these PREs are also recognized by TrxG proteins to antagonise PcG mediated gene repression. In this study, we have characterised DNA binding property of rice trithorax group factor ULTRAPETALA1 (OsULT1) which has a SAND domain and B-box motif. Chromatin immunoprecipitation assay indicates cold induced enrichment of OsULT1 occupancy and a decrease in H3K27me3 mark in the promoter region of OsDREB1b gene, during transcription activation. OsULT1 binds to the cis motif "GAGAG", and the sequence specificity is contributed mainly by the SAND domain. GAGAG is one of the cis motifs present in PREs that are recognized by Drosophila GAGA factor and Pipsqueak. Thus, binding of OsULT1 to GAGAG motif, along with a decrease in H3K27me3 suggests that OsULT1 antagonises the repressive effect of PcG complex for transcriptional activation of OsDREB1b. Moreover, OsULT1 interacts with rice SET domain-containing methyltransferase TRX1, suggesting OsULT1 is an integral part of plant Trithorax group complex. Furthermore, the increase in ULT1 levels during environmental cues suggests its involvement in the transcriptional regulation of stress responsive genes. Collectively, these results suggest that the antagonistic functions of PcG and TrxG proteins and the mechanism of recruitment of these complexes to target loci are evolutionarily conserved for gene expression regulation across kingdoms.

Molecular characterization of the 14-3-3 gene family in rice and its expression studies under abiotic stress.

MAIN CONCLUSION: 14-3-3 isoforms were relatively less conserved at the C-terminal region across plant groups. Both Os 14-3-3f and Os 14-3-3g were inducible with differential gene expression levels under different abiotic stress and developmental stages in sensitive and tolerant indica rice cultivars as confirmed both at transcript and protein level. Plant 14-3-3s has been well characterized to function in several signaling pathways, biotic as well as abiotic stress and nutrient metabolism. We attempted comprehensive analysis of 14-3-3 genes in different plant lineages such as green algae (Chlamydomonas reinhardtii), moss (Physcomitrella patens) and lycophyte (Selaginella moellendorffii), dicot Arabidopsis thaliana and monocot Oryza sativa sub sp. japonica at the gene and protein level. Sequence alignment results revealed that 14-3-3 isoforms were evolutionarily conserved across all taxa with variable C-terminal end. Phylogenetic analysis indicated that the majority of 14-3-3 isoforms in rice belong to the non-epsilon group that clustered separately from the dicot group. Segmental duplication event played a significant role in the expansion of both, Arabidopsis and rice, 14-3-3 isoforms as revealed by synteny studies. In silico gene expression using Massive Parallel Signature Sequencing and microarray analysis revealed that 14-3-3 isoforms have variable expression in different tissue types and under different abiotic stress regime in Arabidopsis and japonica rice. Both, semi-quantitative and qPCR results, confirmed that Os14-3-3f and Os14-3-3g were inducible under abiotic stress in lamina and roots of indica rice and relatively higher under salinity and cold stress in Nonabokra, under dehydration stress in N-22 and under exogenous ABA in IR-29 usually after 3-6 h of treatment. Both, 14-3-3f and 14-3-3g, were highly expressed in flag leaves, stems and panicles and mature roots. These results were further confirmed by immunoblot analysis of rice cultivars using Os14-3-3f antibody generated from recombinant Os14-3-3f protein. The results provide the first comprehensive report of Os14-3-3 gene expression in indica rice cultivars which differ in tolerance to abiotic stress that might be useful for further research.

Understanding the role of DNA polymerase λ gene in different growth and developmental stages of Oryza sativa L. indica rice cultivars.

DNA polymerase λ (Pol λ) is the only member of DNA polymerase family X present in plants. The enzyme is ddNTP sensitive as it contains the conserved C-terminal Pol ß domain. The 1.1 kb partial coding sequence isolated spanned the whole 3' regions of the gene containing functionally important domains of the Pol λ gene. Comparative in silico studies from both indica and japonica cultivars involving homology modelling showed that the model for the partial Pol λ gene was stable and acceptable. The alignment of both the protein models showed a RMS value of 0.783. Apart from this, expression of Pol λ and its relative activity is studied during different development stages of three different indica rice cultivars (IR29, Nonabokra and N22). Enhanced accumulation and higher activity of Pol λ during the early seedling stage was detected. Higher expression and activity were observed in the anthers, which was probably necessary for DNA repair during microspore formation. However, during the maturation stage of seed development and plant growth, expression and the activity of Pol λ decreased due to slow metabolic activity and a reduced rate of cell division respectively. Furthermore, the expression and activity of Pol λ were found to be higher in IR29 in comparison to Nonabokra and N22. IR29 is a rice cultivar susceptible to environmental stresses and hence it encounters higher DNA damages. The enhanced presence and activity of the Pol λ enzyme in IR29 with respect to the other two cultivars, which are more tolerant to the environmental stresses during various developmental stages, is therefore explainable.

Comparative analysis of Histone modifications and DNA methylation at OsBZ8 locus under salinity stress in IR64 and Nonabokra rice varieties.

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region -2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.

Molecular and biochemical characterization of a Vigna mungo MAP kinase associated with Mungbean Yellow Mosaic India Virus infection and deciphering its role in restricting the virus multiplication.

Yellow Mosaic Disease caused by the begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) severely affects many economically important legumes. Recent investigations in Vigna mungo - MYMIV incompatible interaction identified a MAPK homolog in the defense signaling pathway. An important branch of immunity involves phosphorylation by evolutionary conserved Mitogen-activated protein kinases (MAPK) that transduce signals of pathogen invasion to downstream molecules leading to diverse immune responses. However, most of the knowledge of MAPKs is derived from model crops, and functions of these versatile kinases are little explored in legumes. Here we report characterization of a MAP kinase (VmMAPK1), which was induced upon MYMIV-inoculation in resistant V. mungo. Phylogenetic analysis revealed that VmMAPK1 is closely related to other plant-stress-responsive MAPKs. Both mRNA and protein of VmMAPK1 were accumulated upon MYMIV infection. The VmMAPK1 protein localized in the nucleus as well as cytoplasm and possessed phosphorylation activity in vitro. A detailed biochemical characterization of purified recombinant VmMAPK1 demonstrated an intramolecular mechanism of autophosphorylation and self-catalyzed phosphate incorporation on both threonine and tyrosine residues. The Vmax and Km values of recombinant VmMAPK1 for ATP were 6.292nmol/mg/min and 0.7978µM, respectively. Furthermore, the ability of VmMAPK1 to restrict MYMIV multiplication was validated by its ectopic expression in transgenic tobacco. Importantly, overexpression of VmMAPK1 resulted in the considerable upregulation of defense-responsive marker PR genes. Thus, the present data suggests the critical role of VmMAPK1 in suppressing MYMIV multiplication presumably through SA-mediated signaling pathway and inducing PR genes establishing the significant implications in understanding MAP kinase gene function during Vigna-MYMIV interaction; and hence paves the way for introgression of resistance in leguminous crops susceptible to MYMIV.

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana.

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (Kd = 475 ± 17.9 nM) compared to Arabidopsis HMGB1 protein (Kd = 39.8 ± 2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys(85) from the ARID domain and Arg(199) & Lys(202) from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.

Differential acetylation of histone H3 at the regulatory region of OsDREB1b promoter facilitates chromatin remodelling and transcription activation during cold stress.

The rice ortholog of DREB1, OsDREB1b, is transcriptionally induced by cold stress and over-expression of OsDREB1b results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of OsDREB1b is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of OsDREB1b genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼ 700 bp upstream region of OsDREB1b shows two positioned nucleosomes between -610 to -258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of cis elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in OsDREB1b locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of OsDREB1b as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of OsDREB1b gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of OsDREB1b in the transcription "off" state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression.

Functional characterization of a serine-threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fiber formation.

BACKGROUND: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco1128 [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco1128 was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. RESULTS: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T1 tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T0 and T1 plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers. CONCLUSIONS: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant.

Arabinosylated lipoarabinomannan skews Th2 phenotype towards Th1 during Leishmania infection by chromatin modification: involvement of MAPK signaling.

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an anti-inflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal-regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara-LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis.

TLR signaling-mediated differential histone modification at IL-10 and IL-12 promoter region leads to functional impairments in tumor-associated macrophages.

Tumor-associated macrophages (TAM) are severely compromised for the induction of proinflammatory mediators following toll-like receptor (TLR) activation. Here, we reported that the defective TLR response in TAM was due to the malfunctioning of the myeloid differentiation primary response gene 88 (MyD88)-dependent signaling cascade in concert with downregulation of tumor necrosis factor receptor-associated factor (TRAF) 6 and interleukin-1 receptor-associated kinase (IRAK) 1. However, the expression of toll-interleukin1 receptor domain-containing adapter-inducing interferon beta (TRIF) and TRAF 3, which act via the TRIF-dependent pathway of TLR signaling, were found to be unaffected in TAM. Although, TRIF-mediated signal inducers, lipopolysaccharide or poly (I:C), induced high level of extracellular signal-regulated kinase (ERK)-1/2 mitogen-activated protein kinase (MAPK) phosphorylation, but they were failed to induce significant p38MAPK phosphorylation in TAM. Consequently, ERK-1/2-dependent histone phosphorylation at the IL-10 promoter elicited enhanced interleukin (IL)-10 production by TAM. Whereas, the lack of transcription favorable histone phosphorylation at the IL-12 promoter was accompanied with a very low amount of IL-12 expression in TAM. Moreover, ERK-1/2 MAPK activation resulted in enhanced IRAK M induction in TAM, a specific inhibitor of MyD88 pathway. Therefore, for the first time, we decipher an unexplored TLR signaling in TAM where ERK-1/2 activation in a MyD88-independent pathway results in transcription favorable histone modification at the IL-10 promoter region to enhance IL-10-mediated immunosuppression. Additionally, by enhancing IRAK M induction, it also polarizes TAM toward a more immunosuppressive form.

A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants.

Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369-390 with a K(d) of 25 nm, and the other located at residues 28-112 with a K(d) of 160 nm. Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a, and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling.