Extracellular ATP and ectoapyrase in plants
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X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa | Extracellular ATP and ectoapyrase in plants | Scoop.it
Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses in Arabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1- ECD) and that of an orthologous protein, Camelina sativa lectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 A ° resolution from a single crystal. The crystal belonged to space group C222 or C2221, with unit-cell parameters a = 94.7, b = 191.5, c = 302.8 A ° . These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.
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Heptose Sounds the Alarm: Innate Sensing of a Bacterial Sugar Stimulates Immunity

Heptose Sounds the Alarm: Innate Sensing of a Bacterial Sugar Stimulates Immunity | Extracellular ATP and ectoapyrase in plants | Scoop.it
Heptose Is a Sugar Unique to Gram-Negative Bacteria A central tenant to pattern recognition theory is that the structures the host has evolved to interpret as foreign are found solely in the microbial world. Gram-negative bacteria, for example, are defined by the presence of a second membrane outside [...]
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ER-localized adenine nucleotide transporter ER-ANT1: an integrator of energy and stress signaling in rice

ER-localized adenine nucleotide transporter ER-ANT1: an integrator of energy and stress signaling in rice | Extracellular ATP and ectoapyrase in plants | Scoop.it

Most environmental perturbations have a direct or indirect deleterious impact on photosynthesis, and, in consequence, the overall energy status of the cell. Despite our increased understanding of convergent energy and stress signals, the connections between photosynthesis, energy and stress signals through putative common nodes are still unclear. Here we identified an endoplasmic reticulum (ER)-localized adenine nucleotide transporter1 (ER-ANT1), whose deficiency causes seedling lethality in air but viable under high CO2, exhibiting the typical photorespiratory phenotype. Metabolic analysis suggested that depletion of ER-ANT1 resulted in circadian rhythm disorders in sucrose synthesis and induced sucrose signaling pathways, indicating that the ER is involved in the regulation of vital energy metabolism in plants. In addition, the defect of ER-ANT1 triggers ER stress and activates the unfolded protein response in plant cells, suggesting ER stress and photorespiration are closely linked. These findings provide an important evidence for a key role of ER-localized ER-ANT1 in convergent energy and stress signals in rice. Our findings support the idea that ATP is a central signal involved in the plant response to a variety of stresses.

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Light- and temperature-regulated BjAPY2 may have a role in stem expansion of Brassica juncea - Springer

Light- and temperature-regulated BjAPY2 may have a role in stem expansion of Brassica juncea - Springer | Extracellular ATP and ectoapyrase in plants | Scoop.it
Tuber mustard (Brassica juncea (L.) Czern. et Coss. var. tumida Tsen et Lee) is an important vegetable crop with a characteristic of expanded stem that is edible. The underlying molecular mechanism of the stem expansion is not well understood. Here, we reported that a total of 51 differentially expressed fragments (DEFs) with three expression patterns during stem expansion of tuber mustard were identified by cDNA-AFLP analysis. Among the DEFs, DEF11 with high homology to Arabidopsis thaliana apyrase 2 (AtAPY2) that encodes an enzyme with ATPase and ADPase activity was development- and tissue-specific. DEF11 was thus renamed as BjAPY2. The expression levels of BjAPY2 increased with the stem expression and were the highest at stage IV, a developmental stage at which the stem expanded most rapidly. In contrast, the BjAPY2 expression levels in leaves were much lower and remained unchanged during leaf development and expansion, suggesting that BjAPY2 was closely associated with the expansion of stems but not of leaves in the tuber mustard. Interestingly, the expression of BjAPY2 was higher in the mustard under short-day (SD) photoperiod (8 h/16 h) than that under long-day (LD) photoperiod (16 h/8 h); similarly, the transcript levels of BjAPY2 were higher in the mustard grown at low temperature (14 °C/12 °C) than that at high temperature (26 °C /24 °C). The SD photoperiod and low temperature were two environmental conditions that favored the mustard stem expansion. Further cloning and analysis of the promoter region of BjAPY2 revealed that there were indeed several types of motifs in the promoter region, including the light and temperature responsive elements. These results suggested that BjAPY2 might play an important role during the stem expansion of the tuber mustard.
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The Arabidopsis thaliana lectin receptor kinase LecRK-I.9 is required for full resistance to Pseudomonas syringae and affects jasmonate signalling - Molecular Plant Pathology

The Arabidopsis thaliana lectin receptor kinase LecRK-I.9 is required for full resistance to Pseudomonas syringae and affects jasmonate signalling - Molecular Plant Pathology | Extracellular ATP and ectoapyrase in plants | Scoop.it
Upon microbial attack, plants can detect the invaders and activate the plant innate immunity system. For detecting pathogen molecules or cell wall damage, plants employ receptors that trigger the activation of defence responses. Cell surface proteins that belong to large families of lectin receptor kinases are candidates to function as immune receptors. Here the function of LecRK-I.9 (At5g60300), a legume-type lectin receptor kinase involved in cell wall – plasma membrane contacts and in eATP perception, was studied through biochemical, gene expression and reverse genetics approaches. In Arabidopsis thaliana, LecRK-I.9 expression is rapidly, highly and locally induced upon inoculation with avirulent strains of Pseudomonas syringae pv. tomato (Pst). Two allelic lecrk-I.9 knock-out mutants showed decreased resistance to Pst. Conversely, over-expression of LecRK-I.9 led to increased resistance to Pst. Analysis of defence gene expression suggests an alteration of both the salicylic acid (SA) and the jasmonic acid (JA) signalling pathways. In particular, LecRK-I.9 expression during the plant-pathogen interaction was dependent on COI-1 and JAR-1 components and JA-responsive transcription factors (TFs) showed altered levels of expression in plants over-expressing LecRK-I.9. A similar misregulation of these TFs was obtained by JA treatment. This study identified LecRK-I.9 as necessary for full resistance to Pst and pointed out its involvement in the control of defence towards pathogens through a regulation of JA signalling components. The role of LecRK-I.9 is discussed with regard to the potential molecular mechanisms linking JA signalling to cell wall damages and/or eATP perception. This article is protected by copyright. All rights reserved.
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Plant perceptions of extracellular DNA and RNA: Molecular Plant

Plant perceptions of extracellular DNA and RNA: Molecular Plant | Extracellular ATP and ectoapyrase in plants | Scoop.it
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BEEP: An assay to detect bio-energetic and envelope permeability alterations in Pseudomonas aeruginosa

BEEP: An assay to detect bio-energetic and envelope permeability alterations in Pseudomonas aeruginosa | Extracellular ATP and ectoapyrase in plants | Scoop.it
We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased screen of a transposon insertion library in P. aeruginosa strain PAO1 in order to expedite the isolation of mutants with defects in bioenergetic pathways. We successfully isolated insertion mutants that were reduced for extracellular ATP accumulation and identified the corresponding mutations that caused the phenotype. Most of the genes identified from this analysis were associated with energy metabolism and several appeared to be potentially novel bioenergetic targets. In addition, we show that treatment of P. aeruginosa strain PAO1 with antibiotics that disrupt the bacterial cell envelope leads to greater extracellular ATP accumulation. In summary, increases in extracellular ATP accumulation above wild type levels indicated a perturbation of membrane permeability while decreases in extracellular ATP accumulation indicated defects in bioenergetics.
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Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes

Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes | Extracellular ATP and ectoapyrase in plants | Scoop.it
Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26. Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo.
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Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants - New Phytologist 2016

Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants - New Phytologist 2016 | Extracellular ATP and ectoapyrase in plants | Scoop.it
Pattern-triggered immunity (PTI) is a plant defense response that relies on the perception of conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively). Recently, it has been recognized that PTI restricts virus infection in plants; however, the nature of the viral or infection-induced PTI elicitors and the underlying signaling pathways are still unknown. As double-stranded RNAs (dsRNAs) are conserved molecular patterns associated with virus replication, we applied dsRNAs or synthetic dsRNA analogs to Arabidopsis thaliana and investigated PTI responses. We show that in vitro-generated dsRNAs, dsRNAs purified from virus-infected plants and the dsRNA analog polyinosinic–polycytidylic acid (poly(I:C)) induce typical PTI responses dependent on the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1), but independent of dicer-like (DCL) proteins in Arabidopsis. Moreover, dsRNA treatment of Arabidopsis induces SERK1-dependent antiviral resistance. Screening of Arabidopsis wild accessions demonstrates natural variability in dsRNA sensitivity. Our findings suggest that dsRNAs represent genuine PAMPs in plants, which induce a signaling cascade involving SERK1 and a specific dsRNA receptor. The dependence of dsRNA-mediated PTI on SERK1, but not on DCLs, implies that dsRNA-mediated PTI involves membrane-associated processes and operates independently of RNA silencing. dsRNA sensitivity may represent a useful trait to increase antiviral resistance in cultivated plants.
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Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth–Defense Tradeoffs

Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth–Defense Tradeoffs | Extracellular ATP and ectoapyrase in plants | Scoop.it
Plant growth is often constrained by the limited availability of resources in the microenvironment. Despite the continuous threat of attack from insect herbivores and pathogens, investment in defense represents a lost opportunity to expand photosynthetic capacity in leaves and absorption of nutrients and water by roots. To mitigate the metabolic expenditure on defense, plants have evolved inducible defense strategies. The plant hormone jasmonate (JA) is a key regulator of many inducible defenses. Synthesis of JA in response to perceived danger leads to the deployment of a variety of defensive structures and compounds, along with a potent inhibition of growth. Genetic studies have established an important role for JA in mediating tradeoffs between growth and defense. However, several gaps remain in understanding of how JA signaling inhibits growth, either through direct transcriptional control of JA-response genes or crosstalk with other signaling pathways. Here, we highlight recent progress in uncovering the role of JA in controlling growth-defense balance and its relationship to resource acquisition and allocation. We also discuss tradeoffs in the context of the ability of JA to promote increased leaf mass per area (LMA), which is a key indicator of leaf construction costs and leaf life span.
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Shared weapons of blood- and plant-feeding insects: surprising commonalities for manipulating hosts

Shared weapons of blood- and plant-feeding insects: surprising commonalities for manipulating hosts | Extracellular ATP and ectoapyrase in plants | Scoop.it

Insects that reprogram host plants during colonization remind us that the insect side of plant-insect story is just as interesting as the plant side. Insect effectors secreted by the salivary glands play an important role in plant reprogramming. Recent discoveries point to large numbers of salivary effectors being produced by a single herbivore species. Since genetic and functional characterization of effectors is an arduous task, narrowing the field of candidates is useful. We present ideas about types and functions of effectors from research on blood-feeding parasites and their mammalian hosts. Because of their importance for human health, blood-feeding parasites have more tools from genomics and other –omics than plant-feeding parasites. Four themes have emerged: 1) mechanical damage resulting from attack by blood-feeding parasites triggers “early danger signals” in mammalian hosts, which are mediated by eATP, calcium, and hydrogen peroxide, 2) mammalian hosts need to modulate their immune responses to the three “early danger signals” and use apyrases, calreticulins, and peroxiredoxins, respectively, to achieve this, 3) blood-feeding parasites, like their mammalian hosts, rely on some of the same “early danger signals” and modulate their immune responses using the same proteins, and 4) blood-feeding parasites deploy apyrases, calreticulins, and peroxiredoxins in their saliva to manipulate the “danger signals” of their mammalian hosts. We review emerging evidence that plant-feeding insects also interfere with “early danger signals” of their hosts by deploying apyrases, calreticulins and peroxiredoxins in saliva. Given emerging links between these molecules, and plant growth and defense, we propose that these effectors interfere with phytohormone signaling, and therefore have a special importance for gall-inducing and leaf-mining insects, which manipulate host-plants to create better food and shelter.

 
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Light- and temperature-regulated BjAPY2 may have a role in stem expansion of Brassica juncea - Funct Integr Genomics

Light- and temperature-regulated BjAPY2 may have a role in stem expansion of Brassica juncea - Funct Integr Genomics | Extracellular ATP and ectoapyrase in plants | Scoop.it

Tuber mustard (Brassica juncea (L.) Czern. et Coss. var. tumida Tsen et Lee) is an important vegetable crop with a characteristic of expanded stem that is edible. The underlying molecular mechanism of the stem expansion is not well understood. Here, we reported that a total of 51 differentially expressed fragments (DEFs) with three expression patterns during stem expansion of tuber mustard were identified by cDNA-AFLP analysis. Among the DEFs, DEF11 with high homology to Arabidopsis thaliana apyrase 2 (AtAPY2) that encodes an enzyme with ATPase and ADPase activity was development- and tissue-specific. DEF11 was thus renamed as BjAPY2. The expression levels of BjAPY2 increased with the stem expression and were the highest at stage IV, a developmental stage at which the stem expanded most rapidly. In contrast, the BjAPY2 expression levels in leaves were much lower and remained unchanged during leaf development and expansion, suggesting that BjAPY2 was closely associated with the expansion of stems but not of leaves in the tuber mustard. Interestingly, the expression of BjAPY2 was higher in the mustard under short-day (SD) photoperiod (8 h/16 h) than that under long-day (LD) photoperiod (16 h/8 h); similarly, the transcript levels of BjAPY2 were higher in the mustard grown at low temperature (14 °C/12 °C) than that at high temperature (26 °C /24 °C). The SD photoperiod and low temperature were two environmental conditions that favored the mustard stem expansion. Further cloning and analysis of the promoter region of BjAPY2 revealed that there were indeed several types of motifs in the promoter region, including the light and temperature responsive elements. These results suggested that BjAPY2 might play an important role during the stem expansion of the tuber mustard.

 
 
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Role of Ectoapyrases in Nodulation - Chapter 52 - Biological Nitrogen Fixation - Wiley Online Library

Role of Ectoapyrases in Nodulation - Chapter 52 - Biological Nitrogen Fixation - Wiley Online Library | Extracellular ATP and ectoapyrase in plants | Scoop.it
Ectoapyrases are nucleotide hydrolyzing enzymes composed of a transmembrane domain and an extracellular catalytic domain. These enzymes are thought to control the extracellular adenosine triphosphate (ATP) concentration, which is important for plant growth, development, and response to stress. Recent studies have clearly shown that the enzymatic activity of ectoapyrase is essential for nodulation, which is the result of a symbiosis between legume plants and rhizobia resulting in the formation of a specialized organ, the nodule, where biological nitrogen fixation takes place. Ectoapyrase is also required for plant infection by symbiotic arbuscular mycorrhiza, which enhances root nutrient uptake. The available data suggest that ectoapyrases function very early during the establishment of these symbioses, perhaps playing a direct role in plant recognition of the key lipo-chitin signals produced by both rhizobia and mycorrhiza. In this chapter, we summarize recent studies and discuss the function of ectoapyrases in plant–symbiont interactions, with a special focus on the regulation of extracellular nucleotides.
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CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, confers broad-spectrum resistance by activating priming

CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, confers broad-spectrum resistance by activating priming | Extracellular ATP and ectoapyrase in plants | Scoop.it
In Arabidopsis, several L-type lectin receptor kinases (LecRKs) have been identified as putative immune receptors. However, to date, there have been few analyses of LecRKs in crop plants. Virus-induced gene silencing of CaLecRK-S.5 verified the role of CaLecRK-S.5 in broad-spectrum resistance. Compared with control plants, CaLecRK-S.5-silenced plants showed reduced hypersensitive response, reactive oxygen species burst, secondary metabolite production, mitogen-activated protein kinase activation, and defense-related gene expression in response to Tobacco mosaic virus pathotype P0 (TMV-P0) infection. Suppression of CaLecRK-S.5 expression significantly enhanced the susceptibility to Pepper mild mottle virus pathotype P1,2,3, Xanthomonas campestris pv. vesicatoria, Phytophthora capsici, as well as TMV-P0. Additionally, β-aminobutyric acid treatment and a systemic acquired resistance assay revealed that CaLecRK-S.5 is involved in priming of plant immunity. Pre-treatment with β-aminobutyric acid before viral infection restored the reduced disease resistance phenotypes shown in CaLecRK-S.5-silenced plants. Systemic acquired resistance was also abolished in CaLecRK-S.5-silenced plants. Finally, RNA sequencing analysis indicated that CaLecRK-S.5 positively regulates plant immunity at the transcriptional level. Altogether, these results suggest that CaLecRK-S.5-mediated broad-spectrum resistance is associated with the regulation of priming.
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Release of extracellular ATP by bacteria during growth. - PubMed - NCBI

Release of extracellular ATP by bacteria during growth. - PubMed - NCBI | Extracellular ATP and ectoapyrase in plants | Scoop.it
BACKGROUND: Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. RESULTS: We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. CONCLUSION: Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology.
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Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1 | Extracellular ATP and ectoapyrase in plants | Scoop.it
DORN1 ( also known as P2K1 ) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.
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Sources of specificity in plant damaged-self recognition

Sources of specificity in plant damaged-self recognition | Extracellular ATP and ectoapyrase in plants | Scoop.it
Plants perceive injury and herbivore attack via the recognition of damage-associated molecular patterns (DAMPs) and herbivore-associated molecular patterns (HAMPs). Although HAMPs in particular are cues that can indicate the presence of a specific enemy, the application of pure DAMPs or HAMPs frequently activates general downstream responses: membrane depolarization, Ca2+ influxes, oxidative stress, MAPKinase activation and octadecanoid signaling at the molecular level, and the expression of digestion inhibitors, cell wall modifications and other general defenses at the phenotypic level. We discuss the relative benefits of perceiving the non-self versus the damaged-self and of specific versus non-specific responses and suggest that the perception of a complex mixture of DAMPs and HAMPs triggers fine-tuned plant responses. DAMPs such as extracellular ATP (eATP), cell wall fragments, signaling peptides, herbivore-induced volatile organic compounds (HI-VOCs) and eDNA hold the key for a more complete understanding of how plants perceive that and by whom they are attacked.
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Apyrase inhibitors enhance the ability of diverse fungicides to inhibit the growth of different plant pathogenic fungi -- Molecular Plant Pathology

Apyrase inhibitors enhance the ability of diverse fungicides to inhibit the growth of different plant pathogenic fungi -- Molecular Plant Pathology | Extracellular ATP and ectoapyrase in plants | Scoop.it
A previous study demonstrated that treatment of Arabidopsis plants with chemical inhibitors of apyrase enzymes increased their sensitivity to herbicides. In this study we found that the addition of the same or related apyrase inhibitors could potentiate the ability of different fungicides to inhibit the growth of five different pathogenic fungi in plate growth assays. The growth of all five fungi was partially inhibited by three commonly used fungicides, copper octanoate, myclobutanil or propiconazole. However, when these fungicides were individually tested in combination with any one of four different apyrase inhibitors (AI. 1; AI.10; AI. 13; AI. 15), their potency to inhibit the growth of five fungal pathogens was significantly increased compared to when they were applied alone. The apyrase inhibitors were most effective in potentiating the ability of copper octanoate to inhibit fungal growth, and least effective in combination with propiconazole. Among the five pathogens assayed, the one most sensitive to the fungicide-potentiating effects of the inhibitors was Sclerotiana sclerotiorum. Overall, among the 60 treatment combinations tested (5 pathogens, 4 apyrase inhibitors, 3 fungicides), the addition of apyrase inhibitors significantly increased the sensitivity of fungi to the fungicide treatments in 53 of those combinations. Consistent with their predicted mode of action, inhibitors AI.1, AI.10 and AI. 13 each increased the level of propiconazole retained in one of the fungi, suggesting that they could partially block the ability of efflux transporters to remove propiconazole from these fungi. This article is protected by copyright. All rights reserved.
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Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma

Chloroplast-specific in vivo Ca2+ imaging using Yellow Cameleon fluorescent protein sensors reveals organelle-autonomous Ca2+ signatures in the stroma | Extracellular ATP and ectoapyrase in plants | Scoop.it
In eukaryotes, subcellular compartments such as mitochondria, the endoplasmic reticulum, lysosomes and vacuoles have the capacity for Ca2+ transport across their membranes to modulate the activity of compartmentalized enzymes or to convey specific cellular signaling events. In plants, it has been suggested that chloroplasts also display Ca2+ regulation. So far monitoring of stromal Ca2+ dynamics in vivo has exclusively relied on using the luminescent Ca2+ probe aequorin. This technique is, however, limited in resolution and can only provide a readout averaged over chloroplast populations from different cells and tissues. Here we present a toolkit of Arabidopsis Ca2+ sensor lines expressing plastid-targeted FRET-based Yellow Cameleon (YC) sensors. We demonstrate that the probes reliably report in vivo Ca2+ dynamics in the stroma of root plastids in response to extracellular ATP and of leaf mesophyll and guard cell chloroplasts during light-to-low intensity blue light illumination transition. Applying YC sensing of stromal Ca2+ dynamics to single chloroplasts we confirm findings of gradual, sustained stromal Ca2+ increases at the tissue level after light-to-low intensity blue light illumination transitions, but monitor transient Ca2+ spiking as a distinct and previously unknown component of stromal Ca2+ signatures. Spiking was dependent on the availability of cytosolic Ca2+ but not synchronized between the chloroplasts of a cell. In contrast, the gradual sustained Ca2+ increase, occurred independent of cytosolic Ca2+ suggesting intra-organellar Ca2+ release. We demonstrate the capacity of the YC sensor toolkit to identify novel, fundamental facets of chloroplast Ca2+ dynamics and to refine the understanding of plastidial Ca2+ regulation.
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Extracellular ATP: An Essential Apoplastic Messenger in Plants - Springer

Extracellular ATP: An Essential Apoplastic Messenger in Plants - Springer | Extracellular ATP and ectoapyrase in plants | Scoop.it
Adenosine triphosphate (ATP) plays major roles in cell metabolism as an energy supplier and as a substrate for enzymatic reactions. While ATP is well known for its role as an intracellular energy carrier, recent studies have found that ATP exists not only in the cytoplasm, but also in the extracellular matrix. Cytoplasmic ATP can be secreted into the apoplast through wound leakage, secretory vesicles, or transporters in the plasma membrane. As a signaling molecule, extracellular ATP (eATP) regulates plant metabolism, growth and development, and responses to biotic and abiotic stimuli. eATP binds to receptors in the plasma membrane, where it triggers the generation of second messengers, including Ca2+, NO, and reactive oxygen species. These second messengers induce expression of a series of functional genes that promote changes in the cellular structure and physiological function of plant cells. Here, we discuss the progress in research on the function and signaling properties of this novel apoplastic messenger.
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Frontiers -- Apoplastic Nucleoside Accumulation in Arabidopsis Leads to Reduced Photosynthetic Performance and Increased Susceptibility Against Botrytis cinerea

Frontiers -- Apoplastic Nucleoside Accumulation in Arabidopsis Leads to Reduced Photosynthetic Performance and Increased Susceptibility Against Botrytis cinerea | Extracellular ATP and ectoapyrase in plants | Scoop.it

Interactions between plant and pathogen often occur in the extracellular space and especially nucleotides like ATP and NAD have been identified as key players in this scenario. Arabidopsis mutants accumulating nucleosides in the extracellular space were generated and studied with respect to susceptibility against Botrytis cinerea infection and general plant fitness determined as photosynthetic performance. The mutants used are deficient in the main nucleoside uptake system ENT3 and the extracellular nucleoside hydrolase NSH3. When grown on soil but not in hydroponic culture, these plants markedly accumulate adenosine and uridine in leaves. This nucleoside accumulation was accpmpanied by reduced photosystem II efficiency and altered expression of photosynthesis related genes. Moreover, a higher susceptibility towards Botrytis cinerea infection and a reduced induction of pathogen related genes PR1 and WRKY33 was observed. All these effects did not occur in hydroponically grown plants substantiating a contribution of extracellular nucleosides to these effects. Whether reduced general plant fitness, altered pathogen response capability or more direct interactions with the pathogen are responsible for these observations is discussed.

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The plant cell wall as a site for molecular contacts in fungal pathogenesis

The plant cell wall as a site for molecular contacts in fungal pathogenesis | Extracellular ATP and ectoapyrase in plants | Scoop.it

The plant cell wall, the most external layer of the plant surface, is the site where most pathogenic fungi first make contact with host cells. A plant–fungus interaction therefore commences at the interface between the plant and the spore. Our current research focusing on the plant cell wall has discovered an extracellular ecto-nucleoside triphosphate diphosphohydrolase (ecto-NTPDase/apyrase; EC3.6.1.15) as a key player in plant defense before the onset of PTI (PAMP-triggered immunity). This review focuses on our recent findings, especially the role of the plant cell wall in the extracellular defense against fungi as well as fungal strategies resulting in successful infection.

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New Insights in Plant Biology Gained from Research in Space -- Gravitational and Space Research

New Insights in Plant Biology Gained from Research in Space -- Gravitational and Space Research | Extracellular ATP and ectoapyrase in plants | Scoop.it

Recent spaceflight experiments have provided many new insights into the role of gravity in plant growth and development. Scientists have been taking seeds and plants into space for decades in an effort to understand how the stressful environment of space affects them. The resultant data have yielded significant advances in the development of advanced life-support systems for long-duration spaceflight and a better understanding of the fundamental role of gravity in directing the growth and development of plants. Experiments have improved as new spaceflight hardware and technology paved the way for progressively more insightful and rigorous plant research in space. The International Space Station (ISS) has provided an opportunity for scientists to both monitor and control their experiments in real-time. Experiments on the ISS have provided valuable insights into endogenous growth responses, light responses, and transcriptomic and proteomic changes that occur in the microgravity environment. In recent years most studies of plants in space have used Arabidopsis thaliana, but the single-celled, Ceratopteris richardii spore is also a valuable model system that has been used to understand plant gravity response. Experiments using these fern spores have revealed a dynamic and gravity-responsive trans-cell Ca2+ current that directs polarization of these spores and a possible role of extracellular nucleotides in establishing or contributing to this current. As technology continues to improve, spaceflight experiments will provide many new insights into the role and effects of gravity on plant growth and development.

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ATP-induced calcium oscillations in cultured astrocytes

Sample: Cultured hippocampal astrocytes isolated from 18-day-old embryos of SD rats. Loaded with fura-2. Stimulation: Extracellular application of 30 uM ATP....
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Extracellular ATP mediates cellular K+/Na+ homeostasis in two contrasting poplar species under NaCl stress - Trees

Extracellular ATP mediates cellular K+/Na+ homeostasis in two contrasting poplar species under NaCl stress - Trees | Extracellular ATP and ectoapyrase in plants | Scoop.it

Using the non-invasive micro-test technology (NMT), the effects of extracellular ATP (eATP) on salt-altered flux profiles of K+, Na+, and H+ were investigated in salt-tolerant poplar species, Populus euphratica and salt-sensitive P. popularis. A short-term NaCl (100 mM NaCl, 12 h) resulted in a Na+efflux and a correspondingly increased H+ influx in P. euphratica cells, but the effect was not seen inP. popularis. ATP (50 μM) enhanced exchange of Na+ with H+ in salt-stressed cells of two species (6, 12 h), especially in P. popularis. However, the ATP-stimulated Na+ efflux and H+ influx were significantly inhibited by amiloride (a Na+/H+ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H+-ATPase inhibitor), indicating that the ATP induction of Na+ extrusion resulted from an active Na+/H+ antiport across the plasma membrane (PM). NaCl accelerated K+ efflux in the two species, with a more pronounced effect in the salt-sensitive poplar. The salt-induced K+ efflux was markedly restricted by the K+ channel blocker, tetraethylammonium chloride, indicating that the K+efflux is mediated by depolarization-activated outward rectifying K+ channels and non-selective cation channels. ATP benifited poplar cells, especially the salt-sensitive P. popularis, in maintaining K+ homeostasis under external salinity. This was likely the result of activated H+ pump in the PM, which restricted the K+ efflux through the inhibition of depolarization-activated K+ channels in both species. Na+ and K+ flux recordings revealed that non-hydrolysing analogues of ATP, αβ-meATP (50 μM), and ATPγS (50 μM) produced an effect similar to that of the hydrolysable form but with a more pronounced effect. However, ADP- and AMP-stimulated cells (50 μM) exhibited behaviors different from those invoked by ATP, αβ-meATP, and ATPγS treatments. eATP signalling in K+ and Na+homeostasis was blocked by the antagonists of animal P2 receptors, PPADS, and suramin. Moreover, ATP-stimulated Na+ extrusion and reduction of K+ loss in NaCl-stressed cells were inhibited by LaCl3 (an inhibitor of Ca2+-permeable channels) and DPI (an inhibitor of PM NADPH oxidase), indicating that ATP signalling was mediated via second messengers, H2O2 and Ca2+, in the two poplars differing in salt tolerance.

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