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Background Mexico is considered the diversification center for chili species, but these crops are susceptible to infection by pathogens such as Colletotrichum spp., which causes anthracnose disease and postharvest decay in general. Studies have been carried out with isolated strains of Colletotrichum in Capsicum plants; however, under growing conditions, microorganisms generally interact with others, resulting in an increase or decrease of their ability to infect the roots of C. chinense seedlings and thus, cause disease. Results Morphological changes were evident 24 h after inoculation (hai) with the microbial consortium, which consisted primarily of C. ignotum. High levels of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA) were found around 6 hai. These metabolic changes could be correlated with high transcription levels of diacylglycerol-kinase (CchDGK1 and CchDG31) at 3, 6 and 12 hai and also to pathogen gene markers, such as CchPR1 and CchPR5. Conclusions Our data constitute the first evidence for the phospholipids signalling events, specifically DGPP and PA participation in the phospholipase C/DGK (PI-PLC/DGK) pathway, in the response of Capsicum to the consortium, offering new insights on chilis’ defense responses to damping-off diseases.

Phosphatidylinositol transfer protein (PITP) and phosphatidylinositol 4-kinase (PI4K) are very upstream regulatory elements of the phospholipid signaling pathway in the signal transduction network. Unlike in animal systems, their role in stress signaling is poorly understood in plants. To study this area, PITP- and PI4K-overexpressing transgenic barley lines were developed. Morphological and developmental abnormalities were surveyed and characterized. It was revealed that the overexpression of the upstream signaling genes led to more phenotypic abnormalities than in other transgenic studies working with effector genes or even transcription factors. We hypothesize that this high level of abnormalities is the consequence of the modulation of the very upstream signal transduction pathway elements. On the other hand, we also revealed that overexpression of the PITP and PI4K genes increased stress tolerance during hypoxic cold stress, but not during salinity stress. Differences were also found in the level of frost tolerance between the transgenic overexpression plants and the recipient Golden Promise line. Molecular analysis showed that this improvement was not related to the most important cold responsive transcription factors, the CBF genes. We conclude that the transgenic method may be useful to prove the role of an upstream signaling element; however, due to the many developmental consequences that occur as side effects, it is a less advisable approach to achieve improved stress tolerance.

A secreted peptide, a member of a Brassicaceae-specific gene family, acts on pathogen defense response and root development through the phospholipid pathway and ROS regulation., Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner.

Brassica napus was formed through recent hybridization between Brassica oleracea and Brassica rapa and is an important source of edible oil. The genomic characterization of the families of genes involved in oil biosynthesis was undertaken in the B. napus genome assembly to assess the potential impact of selection breeding on gene content and function. We compared oil biosynthesis genes and found that the genes number has a huge difference in 14 different species. There are 2482 homologs in B. napus cv. ZS11 and only 120 homologs in Jatropha curcas. There is a 4.1 fold expansion over Arabidopsis thaliana and >20 fold expansion over J. curcas. However, the distributions of the gene number in the acyl metabolism pathway are highly similar in all the 14 species. The fatty acid elongation and wax biosynthesis pathway, the phospholipid signaling pathway, and the galactolipid, sulfolipid, and phospholipid synthesis pathway are the top pathways in terms of the gene number. A total of 19 positive selection genes were identified in B. napus. Among them, 5 genes are in the phospholipid signaling pathway and 5 genes in the triacylglycerol and fatty acid degradation pathway. These results will help better understand the mechanism and evolution of oil biosynthesis genes.

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.

Chemotaxis is characterized by spontaneous cellular behavior. This spontaneity results, in part, from the stochasticity of intracellular reactions. Spontaneous and random migration of chemotactic cells is regulated by spontaneously generated signals, namely transient local increases in the level of phosphoinositol-3,4,5-triphosphate (PIP3 pulses). In this study, we attempted to elucidate the mechanisms that generate these PIP3 pulses and how the pulses contribute to gradient sensing during chemotaxis. To this end, we constructed a simple biophysical model of intracellular signal transduction consisting of an inositol phospholipid signaling pathway and small GTPases. Our theoretical analysis revealed that an excitable system can emerge from the non-linear dynamics of the model, and that stochastic reactions allow the system to spontaneously become excited, which was corresponded to the PIP3 pulses. Based on these results, we framed a hypothesis of the gradient sensing; a chemical gradient spatially modifies a potential barrier for excitation and then PIP3 pulses are preferentially generated on the side of the cell exposed to the higher chemical concentration. We then validated our hypothesis using stochastic simulations of the signal transduction.

Summary Phospholipid signaling has been recently implicated in diverse cellular processes in higher plants. We identified a cDNA encoding the phospholipase A1 homolog (CaPLA1) from 5-day-old early roots of hot pepper. The deduced amino acid sequence showed that the lipase-specific catalytic triad is well conserved in CaPLA1. In vitro lipase assays and site-directed mutagenesis revealed that CaPLA1 possesses PLA1 activity, which catalyzes the hydrolysis of phospholipids at the sn-1 position. CaPLA1 was selectively expressed in young roots, at days 4–5 after germination, and rapidly declined thereafter, suggesting that the expression of CaPLA1 is subject to control by a development-specific mechanism in roots. Because transgenic work was extremely difficult in hot peppers, in this study we overexpressed CaPLA1 in Arabidopsis so as to provide cellular information on the function of this gene. CaPLA1 overexpressors had significantly longer roots, leaves and petioles, and grew more rapidly than the wild-type plants, leading to an early bolting phenotype with prolonged inflorescence. Microscopic analysis showed that the vegetative tissues of 35S:CaPLA1 plants contained an increased number of small-sized cells, which resulted in highly populated cell layers. In addition, mRNAs for cell cycle-controlled proteins and fatty acid catabolizing enzymes were coordinately upregulated in CaPLA1-overexpressing plants. These results suggest that CaPLA1 is functionally relevant in heterologous Arabidopsis cells, and hence might participate in a subset of positive control mechanisms of cell and tissue growth in transgenic lines. We discuss possible biochemical and cellular functions of CaPLA1 in relation to the phospholipid signaling pathway in hot pepper and transgenic Arabidopsis plants.

Exposure of tri-n-butyl tin chloride (TBTCl) as a stressor to Euglena gracilis Z causes rapid alteration of cell morphology followed by deflagellation. The present study was undertaken to reveal the mechanism of the cell response at a molecular level. Chlamydomonas reinhardtii, in this study E. gracilis Z and its achlorophyllous mutant SM-ZK, gave similar results when subject to the same stressor. Indeed, similar results were obtained with both strains. Next, assuming that the morphological alteration caused by TBTCl is mediated by the inositide phosphate-lipid signaling pathway, the effects of signal transduction and Ca2+ release reagents (mastoparan as a G-protein activator, neomycin as a phospholipase C inhibitor, verapamil as a Ca2+ channel blocker, and A23187 as a Ca2+ ionophore) on morphology and intracellular Ca2+ levels were examined with or without TBTCl. The data strongly suggest that the morphological alteration is mediated by an increase in Ca2+ linked to the inositol phosphatide pathway. The cellular response to signal transduction inducing reagents was compared between the E. gracilis chlorophyllous Z strain and its achlorophyllous mutant SM-ZK strain. Significant differences were observed between the Z and SM-ZK strains in terms of the stress response and intracellular Ca2+ level.

11 pages, 8 figures., In plant tissue, a wound signal is produced at the site of injury and propagates or migrates into adjacent tissue where it induces increased phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity and phenylpropanoid metabolism. We used excised mid-rib leaf tissue from Romaine lettuce (Lactuca sativa L., Longifolia) as a model system to examine the involvement of components of the phospholipid-signaling pathway in wound-induced phenolic metabolism. Exposure to 1-butanol vapors or solutions inhibited wound-induced increase in PAL activity and phenolic metabolism. Phospholipases D (EC 3.1.4.4), an enzyme involved in the phospholipid-signaling pathway is specifically inhibited by 1-butanol. Re-wounding tissue, in which an effective 1-butanol concentration had declined below active levels by evaporation, did not elicit the normal wound response. It appears the 1-butanol-treated tissue developed resistance to wound-induced increases in phenylpropanoid metabolism that persisted even when active levels of 1-butanol were no longer present. However, a metabolic product of 1-butanol, rather than 1-butanol itself, may be the active compound eliciting persistence resistance. Inhibiting a subsequent enzyme in the phospholipid-signaling pathway, lipoxygenase (LOX; EC 1.13.11.12) with 1-phenyl-3-pyrazolidinone (1P3P) or reducing the product of LOX activity with diethyldithio-carbamic acid (DIECA) also inhibited wound-induced PAL activity and phenolic accumulation. The effectiveness of 1-butanol, DIECA, and 1P3P declined as the beginning of the 1-h immersion period was delayed from 0 to 4 h after excision. This decline in effectiveness is consistent with involvement of the inhibitors in the production or propagation of a wound signal. The wound signal in lettuce moves into adjacent tissue at 0.5 cm h−1, so delaying application would allow the signal to move into and induce the wound response in adjacent tissue before the delayed application inhibited synthesis of the signal. Salicylic acid (SA) inhibits allene oxide synthase (AOS, EC 4.2.1.92), another enzyme in the phospholipid-signaling pathway. Exposure to 1 or 10 mM SA for 60 min reduced wound-induced phenolic accumulation by 26 or 56%, respectively. However, 1 mM SA lost its effectiveness if applied 3 h after excision, while 10 mM SA remained effective even when applied 4 h after excision. At 1 mM, SA may be perturbing the wound signal through inhibition of AOS, while at 10 mM it appears to have some generally inhibitory effect on subsequent phenolic metabolism. These data further implicate the phospholipid-signaling pathway in the generation of a wound signal that induces phenolic metabolism in wounded leaf tissue.

The development of nitrogen fixing root nodules is complex and involves an interplay of signaling processes. During maturation of plant host cells and their endocytosed rhizobia in symbiosomes, host cells and symbiosomes expand. This expansion is accompanied by a large quantity of membrane biogenesis. We recently characterized an AGC kinase gene, MtIRE, that could play a role in this expansion. MtIRE's expression coincides with host cell and symbiosome expansion in the proximal side of the invasion zone in developing Medicago truncatula nodules. MtIRE's closest homolog is the Arabidopsis AGC kinase family IRE gene, which regulates root hair elongation. AGC kinases are regulated by phospholipid signaling in animals and fungi as well as in the several instances where they have been studied in plants. Here we suggest that a phospholipid signaling pathway may also activate MtIRE activity and propose possible upstream activators of MtIRE protein's presumed AGC kinase activity.

To know whether or not the set of genes involved in the inositol phospholipid signaling pathway already existed in the early evolution of animals, we carried out cloning of cDNAs encoding phospholipase Cs (PLCs) from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We isolated two PLC cDNAs, PLC-βS and PLC-γS, from sponge and three cDNAs, PLC-βH1, PLC-βH2, and PLC-δH, from hydra. From the domain organization and the divergence pattern in the PLC family tree, the sponge PLC-βS and PLC-γS and the hydra PLC-δH are possibly homologous to the vertebrate PLC-β, PLC-γ and PLC-δ subtypes, respectively. A detailed phylogenetic analysis suggests that the hydra PLC-βH1 and PLC-βH2 are homologs of the vertebrate PLC-β1/2/3/Drosophila PLC21 and the vertebrate PLC-β4/Drosophila norpA, respectively. A phylogenetic analysis of the PLC family and the protein kinase C (PKC) family, together with that of the G protein α subunit (Gα) family, revealed that the origin of the set of genes Gαq, PLC, PKC involved in the inositol phospholipid signaling pathway is very old, going back to dates before the parazoan-eumetazoan split, the earliest branching among extant animal phyla.

Natural killer (NK) cells are important for host defense against malignancy and infection. At a cellular level NK cells are activated when signals from activating receptors exceed signaling from inhibitory receptors. At a molecular level NK cells undergo an education process to both prevent autoimmunity and acquire lytic capacity. Mouse models have shown important roles for inositol phospholipid signaling in lymphocytes. NK cells from mice with deletion in different members of the inositol phospholipid signaling pathway exhibit defects in development, NK cell repertoire expression and effector function. Here we review the current state of knowledge concerning the function of inositol phospholipid signaling components in NK cell biology.

To elucidate the mechanisms of the initracellular signal transduction elicited with bradykinin in NG108-15 neuroblastoma x glioma hybrid cells, we examined the activation of Ca 2+ /calmodulin-dependent protein kinase II (CaM kinase II) by bradykinin stimulation. When the extract of NG108-15 cells was immunoprecipitated with the affinity-purified antibody to brain CaM kinase II, a 50-kDa protein in the immunoprecipitate mainly became autophosphorylated in a Ca 2+ /calmodulin-dependent manner. The result suggest that the 50-kDa protein is the subunit of CaM kinasee II in NG108-15 cells.The Ca 2+ /calmodulin-independent activity (autonomous activity) of the enzyme increased twice within 10 s by stimulation with 1 μM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases: the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 μM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca 2+ with 1 mM EGTA or by the pretreatment with 1 μM nifedipine. Stimulation of 32 P-labeled NG108-15 cells with 1 μM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. Theseesults suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells.

We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells.

Phosphatidylinositol 3 kinases (PI-3 kinases) play a crucial role in the inositol phospholipid signaling pathway. The effect of wortmannin, an inhibitor of PI-3 kinases, on the suppression of thermotolerance development was investigated in the temperature-sensitive cell line tsAF8. When wortmannin was added immediately after the preheating, the thermotolerance was suppressed, with this suppression being dose-dependent from 0.01 to 0.1 microM, but not dose-dependent from 0.1 to 20 microM. Our results suggest that PI-3 kinases might play an important role in thermotolerance development in tsAF8 cells.

Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

Intracellular Ca2+ release activated by inositol 1,4,5-trisphosphate (InsP3) plays a pivotal role in Ca2+ signaling in cells. A controlling mechanism for InsP3-induced Ca2+ movements is suggested by results showing that the InsP3-releasable Ca2+ pool is directly modified by a specific and sensitive GTP-regulated Ca2+-translocating process. By using saponin-permeabilized N1E-115 neuroblastoma cells or DDT1MF-2 smooth muscle-derived cells, InsP3 releases 30-50% of Ca2+ accumulated through intracellular high-affinity ATP-dependent Ca2+-pumping activity. Oxalate-promoted Ca2+ uptake is reversed by InsP3, indicating oxalate permeability of the InsP3-releasable pool, which is consistent with this compartment being the endoplasmic reticulum. GTP (10 microM) activates release of 50-70% of accumulated Ca2+ from cells. In the presence of 5-10 mM oxalate, GTP induces a biphasic Ca2+ flux response; initially (1-2 min) GTP induces rapid Ca2+ release followed thereafter by a profound increase in Ca2+ uptake. Thus, GTP-activated Ca2+ influx and efflux compete for Ca2+ access to the oxalate-permeable Ca2+ pool. The nonadditive effects of InsP3 and GTP suggest that InsP3 releases Ca2+ from a subcompartment of the GTP-releasable pool. Most significantly, InsP3 is observed to block the GTP-activated uptake phase in the presence of oxalate, indicating that GTP induces Ca2+ entry into the pool from which InsP3 activates release. Hence, the results provide direct evidence that loading of Ca2+ into the InsP3-sensitive Ca2+ pool is controlled by a GTP-regulated Ca2+-translocating mechanism. Such a process could be significant in regulating the extent and duration of the InsP3-induced Ca2+ signal, a crucial step in the inositol phospholipid signaling pathway.