Extracellular ATP and ectoapyrase in plants
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Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture

Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture | Extracellular ATP and ectoapyrase in plants | Scoop.it
In addition to acting as a cellular energy source, ATP can also act as a damage-associated molecular pattern in both animals and plants. Stomata are leaf pores that control gas exchange and, therefore, impact critical functions such as photosynthesis, drought tolerance, and also are the preferred entry point for pathogens. Here we show the addition of ATP leads to the rapid closure of leaf stomata and enhanced resistance to the bacterial pathogen Psuedomonas syringae. This response is mediated by ATP recognition by the receptor DORN1, followed by direct phosphorylation of the NADPH oxidase RBOHD, resulting in elevated production of reactive oxygen species and stomatal closure. Mutation of DORN1 phosphorylation sites on RBOHD eliminates the ability of ATP to induce stomatal closure. The data implicate purinergic signaling via DORN1 in the control of stomatal aperture with important implications for the control of plant photosynthesis, water homeostasis, pathogen resistance, and ultimately yield.

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Two-dimensional array ATP/ADP sensitive image sensor with a uniform distribution of chemically immobilized Apyrase | Bulletin of the Chemical Society of Japan | CSJ Journals

Two-dimensional array ATP/ADP sensitive image sensor with a uniform distribution of chemically immobilized Apyrase | Bulletin of the Chemical Society of Japan | CSJ Journals | Extracellular ATP and ectoapyrase in plants | Scoop.it
ATP and ADP are the major energy source in metabolism of cells, still more ATP works as an important extracellular signaling material. ATP bioimaging is required to understand their metabolism. In this paper, a label-free ATP/ADP image sensor was fabricated using a 128×128 (16 k) pixels array semiconductor CCD-type pH image sensor and Apyrase. The principle is based on measuring proton produced by the enzyme reaction between Apyrase and ATP or ADP. In order to put a uniform potential response on the sensor into practice, two different methods (3-APTES and CEST) which chemically fixed N-terminal of Apyrase with the sensor were examined. The sensor modified by the CEST method had a quite clean surface microscopically and demonstrated a fine real-time image of monitoring the ATP concentration. The potential response of the image sensor was characterized; effect of buffer solution, calibration curves of ATP and ADP, durability, the limit of detection (LOD) for ATP, and the response of time. The potential distribution for effective pixels to ATP concentration was narrow, single and symmetry. Due to 16 K sensing pixels finely responded to ATP concentration uniformly, the LOD of ATP concentration (10 μM) was exactly determined with a statistical treatment.

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Wounding triggers callus formation via dynamic hormonal and transcriptional changes

Wounding triggers callus formation via dynamic hormonal and transcriptional changes | Extracellular ATP and ectoapyrase in plants | Scoop.it
Wounding is a primary trigger of organ regeneration, but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study, we combined transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis thaliana (Arabidopsis). Our time-course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes, starting from rapid stress responses followed by the activation of metabolic processes and protein synthesis and subsequent activation of cell cycle regulators. Gene ontology analyses further uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signaling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin is critical for wound-induced callus formation. We further demonstrate that type-B ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signaling regulates the expression of CYCLIN D3;1 (CYCD3;1) and that mutations in CYCD3;1 and its homologs CYCD3;2-3 cause defects in callus formation. In addition to these hormone-mediated changes, our transcriptome data uncovered that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 (ERF115) and PLETHORA3 (PLT3), PLT5, PLT7 in callus generation. Altogether, these results provide novel mechanistic insights into how wounding reactivates cell proliferation during callus formation.
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The responses of photosystem II and intracellular ATP production of Arabidopsis leaves to salt stress are affected by extracellular ATP

The responses of photosystem II and intracellular ATP production of Arabidopsis leaves to salt stress are affected by extracellular ATP | Extracellular ATP and ectoapyrase in plants | Scoop.it

Hypertonic salt stress with different concentrations of NaCl increased the levels of extracellular ATP of Arabidopsis leaves. And, hypertonic salt stress decreased the levels of Fv/Fm (the maximal efficiency of photosystem II), ΦPSII (the photosystem II operating efficiency), qP (photochemical quenching), and intracellular ATP (iATP) production. The treatment with β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP), which can exclude extracellular ATP from its binding sites of extracellular ATP receptors, caused a further decrease in the levels of Fv/Fm, ΦPSII,qP, and iATP production of the salt-stressed Arabidopsis leaves, while the addition of exogenous ATP rescued the inhibitory effects of AMP-PCP on ΦPSII, qP, and iATP production under hypertonic salt stress. Under hypertonic salt stress, the values of Fv/Fm, ΦPSII, qP, and iATP production were lower in the dorn 1–3 mutant than in the wild-type plants. These results indicate that the responses of photosystem II and intracellular ATP production to salt stress could be affected by extracellular ATP.

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Frontiers | Heterotrimeric G Protein-Regulated Ca2+ Influx and PIN2 Asymmetric Distribution Are Involved in Arabidopsis thaliana Roots' Avoidance Response to Extracellular ATP

Frontiers | Heterotrimeric G Protein-Regulated Ca2+ Influx and PIN2 Asymmetric Distribution Are Involved in Arabidopsis thaliana Roots' Avoidance Response to Extracellular ATP | Extracellular ATP and ectoapyrase in plants | Scoop.it
Extracellular ATP (eATP) has been reported to be involved in plant growth as a primary messenger in the apoplast. Here, roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP, showing a marked avoidance response. Roots responded similarly to ADP and bz-ATP but did not respond to AMP and GTP. The eATP avoidance response was reduced in loss-of-function mutants of heterotrimeric G protein α subunit (Gα) (gpa1-1 and gpa1-2) and enhanced in Gα-over-expression (OE) lines (wGα and cGα). EGTA and Gd3+ remarkably suppressed eATP-induced root bending. ATP-stimulated Ca2+ influx was impaired in Gα null mutants and increased in its OE lines. DR5-GFP and PIN2 were asymmetrically distributed in ATP-stimulated root tips, this effect was strongly suppressed by EGTA and diminished in Gα null mutants. In addition, some eATP-induced genes’ expression was also impaired in Gα null mutants. Based on these results, we propose that heterotrimeric Gα-regulated Ca2+ influx and PIN2 distribution may be key signaling events in eATP sensing and avoidance response in Arabidopsis thaliana roots.
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L-type lectin receptor kinases: New forces in plant immunity

L-type lectin receptor kinases: New forces in plant immunity | Extracellular ATP and ectoapyrase in plants | Scoop.it

To halt pathogen invasion, plants—unlike animals—solely depend on an innate immune system. They possess an expanded arsenal of cell surface-localized pattern recognition receptors (PRRs) to perceive microbe- and damage-associated molecular patterns (M/DAMPs), here collectively termed invasions patterns [1]. Prominent examples are flagellin sensing 2 (FLS2) and elongation factor Tu (EF-Tu) receptor (EFR), 2 receptor-like kinases (RLKs) with leucine-rich repeat (LRR) ectodomains that initiate defense upon recognition of bacterial flagellin and EF-Tu, respectively [2]. Emerging key players in plant immunity are the lectin receptor kinases, RLKs that are subdivided in 3 distinct classes based on their extracellular lectin domains, i.e., G- (GNA-related or S-locus), C- (calcium-dependent), and L- (legume) type [3]. All 3 are omnipresent in plants but absent in animals, which deploy distinct C-type lectin receptors (CLRs) to initiate innate immunity [4]. Recent years have witnessed an accelerated interest in plant lectin receptors kinases. In this Pearl, we summarize our current knowledge on L-type lectin receptor kinases (LecRKs) in plant immunity.

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The Evolution of Calcium-Based Signalling in Plants

The Evolution of Calcium-Based Signalling in Plants | Extracellular ATP and ectoapyrase in plants | Scoop.it

The calcium-based intracellular signalling system is used ubiquitously to couple extracellular stimuli to their characteristic intracellular responses. It is becoming clear from genomic and physiological investigations that while the basic elements in the toolkit are common between plants and animals, evolution has acted in such a way that, in plants, some components have diversified with respect to their animal counterparts, while others have either been lost or have never evolved in the plant lineages. In comparison with animals, in plants there appears to have been a loss of diversity in calcium-influx mechanisms at the plasma membrane. However, the evolution of the calcium-storing vacuole may provide plants with additional possibilities for regulating calcium influx into the cytosol. Among the proteins that are involved in sensing and responding to increases in calcium, plants possess specific decoder proteins that are absent from the animal lineage. In seeking to understand the selection pressures that shaped the plant calcium-signalling toolkit, we consider the evolution of fast electrical signalling. We also note that, in contrast to animals, plants apparently do not make extensive use of cyclic-nucleotide-based signalling. It is possible that reliance on a single intracellular second-messenger-based system, coupled with the requirement to adapt to changing environmental conditions, has helped to define the diversity of components found in the extant plant calcium-signalling toolkit.


Via Jean-Michel Ané
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ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology | Extracellular ATP and ectoapyrase in plants | Scoop.it

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

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Calcium signatures and signaling events orchestrate plant–microbe interactions - ScienceDirect

Calcium signatures and signaling events orchestrate plant–microbe interactions - ScienceDirect | Extracellular ATP and ectoapyrase in plants | Scoop.it
Calcium (Ca2+) acts as an essential second messenger connecting the perception of microbe signals to the establishment of appropriate immune and symbiotic responses in plants. Accumulating evidence suggests that plants distinguish different microorganisms through plasma membrane-localized pattern recognition receptors. The particular recognition events are encoded into Ca2+ signatures, which are sensed by diverse intracellular Ca2+ binding proteins. The Ca2+ signatures are eventually decoded to distinct downstream responses through transcriptional reprogramming of the defense or symbiosis-related genes. Recent observations further reveal that Ca2+-mediated signaling is also involved in negative regulation of plant immunity. This review is intended as an overview of Ca2+ signaling during immunity and symbiosis, including Ca2+ responses in the nucleus and cytosol.
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Plant Lectins and Lectin Receptor-Like Kinases: How Do They Sense the Outside?

Plant Lectins and Lectin Receptor-Like Kinases: How Do They Sense the Outside? | Extracellular ATP and ectoapyrase in plants | Scoop.it
Lectins are fundamental to plant life and have important roles in cell-to-cell communication; development and defence strategies. At the cell surface; lectins are present both as soluble proteins (LecPs) and as chimeric proteins: lectins are then the extracellular domains of receptor-like kinases (LecRLKs) and receptor-like proteins (LecRLPs). In this review; we first describe the domain architectures of proteins harbouring G-type; L-type; LysM and malectin carbohydrate-binding domains. We then focus on the functions of LecPs; LecRLKs and LecRLPs referring to the biological processes they are involved in and to the ligands they recognize. Together; LecPs; LecRLKs and LecRLPs constitute versatile recognition systems at the cell surface contributing to the detection of symbionts and pathogens; and/or involved in monitoring of the cell wall structure and cell growth.
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 Extracellular Alkalinization as a Defense Response in Potato Cells 

 Extracellular Alkalinization as a Defense Response in Potato Cells  | Extracellular ATP and ectoapyrase in plants | Scoop.it
A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists (Phytophthora infestans and Spongospora subterranea) and fungi (Verticillium dahliae and Colletotrichum coccodes). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.
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Physiological characterization of a plant mitochondrial calcium uniporter in vitro and in vivo

Physiological characterization of a plant mitochondrial calcium uniporter in vitro and in vivo | Extracellular ATP and ectoapyrase in plants | Scoop.it
Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red (RR) and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC and identifies new player of the intracellular Ca2+ regulation network 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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A high-sensitivity, microtiter-based plate assay for plant MAMP-triggered immunity | Molecular Plant-Microbe Interactions

A high-sensitivity, microtiter-based plate assay for plant MAMP-triggered immunity | Molecular Plant-Microbe Interactions | Extracellular ATP and ectoapyrase in plants | Scoop.it

The first step in the plant immune response to pathogen challenge involves the perception of conserved epitopes, called microbe-associated molecular patterns (MAMPs), by cell surface pattern recognition receptors (PRRs). Given the key roles that MAMPs and PRRs play in plant innate immunity, great effort has been expended to identify these molecules. Current methods for assaying these immune responses are often limited in their resolution and throughput, and consequently, there is a need for medium- to high-throughput methodologies. Here, we describe the development of a 96-well microtiter plate-based assay for plant PTI that measures the activity of plant peroxidase (POX) enzymes produced in response to treatment with bacterial MAMPs. The system has been optimized to minimize both the amount of plant tissue and MAMP required, and displays up to three orders of magnitude greater sensitivity than the traditional luminol-based reactive oxygen species (ROS) assay when measuring the plant response to treatment with the bacterial MAMP flg22, reaching detection limits in the picomolar range. This high sensitivity opens up the possibility of evaluating the immune-eliciting effects of weaker elicitors. The throughput and material requirements of the assay make it ideal for screens involving quantitative measurement of the plant innate immune response to MAMPs.

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Extracellular ATP acts on jasmonate signaling to reinforce plant defense

Extracellular ATP acts on jasmonate signaling to reinforce plant defense | Extracellular ATP and ectoapyrase in plants | Scoop.it
Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea. The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1-3. Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers, cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1, but not in the JA biosynthesis mutant, aos, or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct crosstalk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses.
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Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor | Extracellular ATP and ectoapyrase in plants | Scoop.it

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.

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Molecular Mechanism of Plant Recognition of Extracellular ATP

Molecular Mechanism of Plant Recognition of Extracellular ATP | Extracellular ATP and ectoapyrase in plants | Scoop.it

Adenosine 5′-triphosphate (ATP), a ubiquitously dispersed biomolecule, is not only a major source of biochemical energy for living cells, but also acts as a critical signaling molecule through inter-cellular communication. Recent studies have clearly shown that extracellular ATP is involved in various physiological processes in plants, including root growth, stomata movement, pollen tube development, gravitropism, and abiotic/biotic stress responses. The first plant purinergic receptor for extracellular ATP, DORN1 (the founding member of the P2K family of purinergic receptors), was identified in Arabidopsis thaliana by a forward genetic screen. DORN1 consists of an extracellular lectin domain, transmembrane domain, and serine/threonine kinase, intracellular domain. The predicted structure of the DORN1 extracellular domain revealed putative key ATP binding residues but an apparent lack of sugar binding. In this chapter, we summarize recent studies on the molecular mechanism of plant recognition of extracellular ATP with specific reference to the role of DORN1.

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Multiparameter imaging of calcium and abscisic acid and high‐resolution quantitative calcium measurements using R‐GECO1‐mTurquoise in Arabidopsis

Multiparameter imaging of calcium and abscisic acid and high‐resolution quantitative calcium measurements using R‐GECO1‐mTurquoise in Arabidopsis | Extracellular ATP and ectoapyrase in plants | Scoop.it
Calcium signals occur in specific spatio-temporal patterns in response to various stimuli and are coordinated with, for example, hormonal signals, for physiological and developmental adaptations. Quantification of calcium together with other signalling molecules is required for correlative analyses and to decipher downstream calcium-decoding mechanisms. Simultaneous in vivo imaging of calcium and abscisic acid has been performed here to investigate the interdependence of the respective signalling processes in Arabidopsis thaliana roots. Advanced ratiometric genetically encoded calcium indicators have been generated and in vivo calcium calibration protocols were established to determine absolute calcium concentration changes in response to auxin and ATP. In roots, abscisic acid induced long-term basal calcium concentration increases, while auxin triggered rapid signals in the elongation zone. The advanced ratiometric calcium indicator R-GECO1-mTurquoise exhibited an increased calcium signal resolution compared to commonly used Förster resonance energy transfer-based indicators. Quantitative calcium measurements in Arabidopsis root tips using R-GECO1-mTurquoise revealed detailed maps of absolute calcium concentration changes in response to auxin and ATP. Calcium calibration protocols using R-GECO1-mTurquoise enabled high-resolution quantitative imaging of resting cytosolic calcium concentrations and their dynamic changes that revealed distinct hormonal and ATP responses in roots.

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Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism | Extracellular ATP and ectoapyrase in plants | Scoop.it

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a “butterfly-like” motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.

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A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana

A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana | Extracellular ATP and ectoapyrase in plants | Scoop.it

Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants.

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The Cyclic Nucleotide-Gated Channel CNGC14 Regulates Root Gravitropism in Arabidopsis thaliana - ScienceDirect

The Cyclic Nucleotide-Gated Channel CNGC14 Regulates Root Gravitropism in Arabidopsis thaliana - ScienceDirect | Extracellular ATP and ectoapyrase in plants | Scoop.it
In plant roots, auxin inhibits cell expansion, and an increase in cellular auxin levels on the lower flanks of gravistimulated roots suppresses growth and thereby causes downward bending. These fundamental features of root growth responses to auxin were first described over 80 years ago [1], but our understanding of the underlying molecular mechanisms has remained scant. Here, we report that CYCLIC NUCLEOTIDE-GATED CHANNEL 14 (CNGC14) is essential for the earliest phase of auxin-induced ion signaling and growth inhibition in Arabidopsis roots. Using a fluorescence-imaging-based genetic screen, we found that cngc14 mutants exhibit a complete loss of rapid Ca2+ and pH signaling in response to auxin treatment. Similarly impaired ion signaling was observed upon gravistimulation. We further developed a kinematic analysis approach to study dynamic root growth responses to auxin at high spatiotemporal resolution. These analyses revealed that auxin-induced growth inhibition and gravitropic bending are significantly delayed in cngc14 compared to wild-type roots, where auxin suppresses cell expansion within 1 min of treatment. Finally, we demonstrate that auxin-induced cytosolic Ca2+ changes are required for rapid growth inhibition. Our results support a direct role for CNGC14-dependent Ca2+ signaling in regulating the early posttranscriptional phase of auxin growth responses in Arabidopsis roots.
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An energetic molecule DAMPens plant disease by boosting defence mechanisms

An energetic molecule DAMPens plant disease by boosting defence mechanisms | Extracellular ATP and ectoapyrase in plants | Scoop.it

How do plants, which are, by definition, rooted to the spot, identify and respond to potential threats?

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Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species | Plant and Cell Physiology | Oxford Academic

Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species | Plant and Cell Physiology | Oxford Academic | Extracellular ATP and ectoapyrase in plants | Scoop.it
Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.
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Ectopic expression of Arabidopsis L-type lectin receptor kinase genes LecRK-I.9 and LecRK-IX.1 in Nicotiana benthamiana confers Phytophthora resistance

Ectopic expression of Arabidopsis L-type lectin receptor kinase genes LecRK-I.9 and LecRK-IX.1 in Nicotiana benthamiana confers Phytophthora resistance | Extracellular ATP and ectoapyrase in plants | Scoop.it
In plants, cell surface receptors mediate the first layer of innate immunity against pathogenic microbes. In Arabidopsis several L-type lectin receptor kinases (LecRKs) were previously found to function as Phytophthora resistance components. In this study, we determined the functionality of Arabidopsis LecRK-I.9 or LecRK-IX.1 in Phytophthora resistance when transferred into the Solanaceous plant Nicotiana benthamiana. Multiple transgenic lines were generated for each LecRK gene and molecular analyses revealed variation in transgene copy number, transgene expression levels and LecRK protein accumulation. Infection assays showed that transgenic N. benthamiana plants expressing either Arabidopsis LecRK-I.9 or LecRK-IX.1 are more resistant to Phytophthora capsici and to Phytophthora infestans. These results demonstrate that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their Phytophthora resistance function when transferred into N. benthamiana. Therefore, these LecRKs have the potential to function as a complementary Phytophthora resistance resource in distantly related plant species next to the canonical Phytophthora resistance genes encoding nucleotide-binding leucine-rich repeat proteins.
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Exogenous ATP enhance signal response of suspension cells of transgenic rice (Oryza sativa L.) expressing maize C4-pepc encoded phosphoenolpyruvate carboxylase under PEG treatment

Exogenous ATP enhance signal response of suspension cells of transgenic rice (Oryza sativa L.) expressing maize C4-pepc encoded phosphoenolpyruvate carboxylase under PEG treatment | Extracellular ATP and ectoapyrase in plants | Scoop.it
The aim of this study was to analyze the biochemical and molecular responses of suspension cells of transgenic rice (Oryza sativa L.) over-expressing the maize C4-pepc gene encodes phosphoenolpyruvate carboxylase (PEPC) (PC line) to drought stress by exogenous ATP. Suspension cells of PC and wild-type (WT) rice cultured in N6 basic medium were subjected to the following treatments: a 24-h drought treatment imposed by 20% (m/v) polyethylene glycol 6000 (PEG6000 treatment); an exogenous adenosine 5′-triphosphate treatment (ATP; 1 mmol L−1); and a combined exogenous ATP + PEG6000 treatment. We measured DNA fragmentation, cell viability, lipid peroxidation, antioxidant enzyme activities, PEPC activity, ATP content, and the levels of Ca2+, H2O2, and NO using spectrophotometric methods and fluorescent dyes, and quantified the transcript levels of stress-related genes such as NAC1 (one of the transcription factors from NAC (NAM/ATAF/CUC) family), cytochrome P450 (P450) and programmed cell death (PCD) under these treatments. Compared with WT cells, PC cells showed more stable cellular activity, lower lipid peroxidation, enhanced antioxidant enzyme activity, higher levels of endogenous ATP level, PEPC activity, and transcript levels of C4-pepc and the transcription factor NAC1 under drought stress. While the content of H2O2 and the transcript levels of P450 and PCD were decreased and kept at the low levels during the same treatments. Adding ATP, PC reinforced these early signaling effects response to PEG6000 treatment. Based on the results, the exogenous ATP triggers a series of signaling cascade reactions for enhancing antioxidant protection in PC cells during the drought treatment, conferring to the drought-tolerance.
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