462 research outputs found
In Vivo Analysis of Calcium Levels and Glutathione Redox Status in Arabidopsis Epidermal Leaf Cells Infected with the Hypersensitive Response-Inducing Bacteria Pseudomonas syringae pv. tomato AvrB (PstAvrB)
No full textPlants react to the attack of pathogen microorganisms by mounting appropriate and efficient downstream defense responses often involving a form of localized cell death called hypersensitive response (HR).Here we describe an innovative and noninvasive protocol based on in vivo bioimaging technique coupled with utilization of genetically encoded fluorescent sensors that allows to monitor and analyze intracellular calcium (Ca2+) dynamics and changes of the glutathione redox status taking place in plant organs during plant interaction with the HR-inducing bacteria Pseudomonas syringae (PstAvrB)
TWO CLADE III GLUTAMATE RECEPTOR-LIKE ISOFORMS INVERSELY REGULATE LOCAL AND LONG-DISTANCE CA2+ SIGNALLING IN ARABIDOPSIS THALIANA
No full textIn the central nervous system, ionotropic Glutamate Receptors (iGluRs) are tetrameric ligand-gated non-selective cation channels. They have been linked to learning and memory as well as to neurodegenerative pathologies such as Alzheimer disease. Thus, they are among the best characterized channels in animals. Animals and plants share this class of proteins. Plant Glutamate Receptors-like (GLRs) have been implicated in stomata movement regulation, pollen tube growth, long-distance signalling, root development and defence against pathogens. However, to date few details are known about their basic properties and functions, such as binding activity, ion transportation, sub-cellular localization, subunits interaction, desensitization etc. My PhD project focused on two GLR isoforms in Arabidopsis thaliana, AtGLR3.3 and AtGLR3.7 (hereafter called ‘AtGLR3.x’). Being putative amino acids-gated Ca2+-permeable channels, I tested the hypothesis that the two isoforms could handle Ca2+ dynamics upon amino acids challenge. Remarkably, Arabidopsis Col-0 plants show a transient elevation of cytosolic Ca2+ at the root tip meristematic zone upon amino acids treatment. Ablation of the AtGLR3.3 abolished the increase of Ca2+ concentration whereas loss of-function mutants for AtGLR3.7 showed enhanced Ca2+ rises in response to amino acids. Additionally, when the double mutant glr3.3glr3.7 was challenged with amino acids, mirrored the glr3.3 null-response. These results strongly suggested that the two AtGLRs could assemble in a channel where GLR3.3 would act as main scaffold and GLR3.7 would negatively regulate the biophysical properties. Being AtGLR3.x also expressed in the vascular tissues and in the cells of the floral abscission zone, we also assayed the role of AtGLR3.x in the generation/propagation of long-distance Ca2+ waves that occurs between stems and inflorescence apexes of Arabidopsis plants subjected to flaming.
In the future, our efforts will be aimed at understanding whether the predicted amino acid sensing of GLRs is also required for the long-distance signalization
Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana
No full textCalcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae and algal rhizoids). High-resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that light sheet fluorescence microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosol-localized ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterized their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs
Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots.
No full textPhosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca2+ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca2+ ([Ca2+]cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca2+]cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca2+]cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca2+]cyt, Pi starvation strongly affects stress-induced [Ca2+]cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer
Endoplasmic reticulum-localized CCX2 is required for osmotolerance by regulating ER and cytosolic Ca2+ dynamics in Arabidopsis
No full textCa2+ signals in plant cells are important for adaptive responsesto environmental stresses. Here, we report that the ArabidopsisCATION/Ca2+ EXCHANGER2 (CCX2), encoding a putative novelcation/Ca2+ exchanger that localizes to the endoplasmic reticulum,is strongly induced by salt and osmotic stresses. Compared withthe wild type, CCX2 loss- and gain-of-function mutants were lessand more tolerant to different osmotic stress, respectively, anddisplayed the most noteworthy phenotypes during salt stress.Remarkably, Cameleon Ca2+ sensors revealed that CCX2 activityimpacts cytosolic and Endoplasmic Reticulum (ER) Ca2+ concentrations.To our knowledge, ccx2 is the first plant mutant with ameasured alteration in ER Ca2+ concentrations. In this study, weidentified CCX2 as a pivotal player in the regulation of ER Ca2+dynamics that heavily impinge on plant growth upon salt stress.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
In Vivo Light Sheet Fluorescence Microscopy of Calcium Oscillations in Arabidopsis thaliana
No full textCalcium imaging in plants requires a high-resolution microscope, able to perform volumetric acquisition in a few seconds, inducing as low photobleaching and phototoxicity as possible to the sample. Light sheet fluorescence microscopy offers these capabilities, with the further chance to mount the sample in vertical position, mimicking the plant's growth and physiological conditions.A protocol for plant preparation and mounting in a light sheet microscope is presented. First, the growth of Arabidopsis thaliana in a sample holder compatible with light sheet microscopy is described. Then, the requirements for sample alignment and image acquisition are detailed. Finally, the image processing steps to analyze calcium oscillations are discussed, with particular emphasis on ratiometric calcium imaging in Arabidopsis root hairs
Stochastic simulation of non-stationary continuous multifractal time series
No full textIntermittency is an ubiquitous property of fully developed turbulence, for Eulerian and Lagrangian fields, and for velocity, passive and active scalars. Intermittency corresponds to multi-scale high fluctuations, with some underlying long-range correlations. Such property is usually characterized using scaling approaches, verified using experimental or numerical data. However there are only few studies devoted to the generation of continuous stochastic processes having non-stationary multifractal properties, able to mimic Eulerian or Lagrangian velocity or passive scalar time series. Here we review recent works on this topic, and we provide stochastic simulations in order to verify the theoretical predictions. In the lognormal framework we provide a plane expressing the scale invariant properties of these simulations
Three-component velocity measurements in a momentum-conserving, axisymmetric, turbulent jet
No full textExperiments have been performed on a momentum conserving axisymmetric turbulent jet, the turbulence characteristics of which are well known [1]. Simultaneous three-component velocity measurements are acquired with high spatial and temporal resolution, using a new triple-sensor hotwire probe. Velocity and directional calibrations are performed using a dedicated automatic calibration system. Two experiments are performed; one for capturing the average velocity field in a 3D volume, and one for investigating the turbulence spectra in specific points in space. In the first experiment, measurements are performed in 9 equidistant cross-planes, from 10- to 50-diameters downstream of the nozzle using a computer-controlled traversing system. The spatial resolution is as low as 1 mm and the sampling rate was 10 kHz. In the second experiment, long velocity time histories are acquired with 50 kHz sampling rate to perform power spectral density computations for each velocity component. Preliminary results of velocity capture confirm the general characteristics of the turbulent jet. The power spectra at different positions indicate that the turbulent fluctuations are not isotropic at lower frequencies
Joint scaling analysis of atmospheric velocity and wind power plant production
No full textIn a context of energy transition, wind energy is a source of clean energy with the potential of partially satisfying the growing demand. The main problem of this type of energy, and other types of renewable energy remains the discontinuity of the electric power produced in different scales, inducing large fluctuations also called intermittency. This intermittency of wind energy is inherent to the turbulent nature of wind. Here, we consider the relation between velocity and power output with two wind turbine databases. We focus on joint relations with Fourrier analysis, empirical mode decomposition (EMD), Time-dependent intrinsic correlation (TDIC). We also consider the causality using a new method of analysis of the causality between two time series
Cellular Ca2+ signals generate defined pH signatures in plants
No full textCalcium ions (Ca2+) play a key role in cell signaling across organisms. The question of how a simple ion can mediate specificity has spurred research into the role of Ca2+ signatures and their encoding and decoding machinery. Such studies have frequently focussed on Ca2+ alone and our understanding of how Ca2+ signalling is integrated with other responses remains poorly understood. Using in vivo imaging with different genetically-encoded fluorescent sensors in Arabidopsis cells we show that Ca2+ transients do not occur in isolation but are accompanied by pH changes in the cytosol. We estimate the degree of cytosolic acidification at up to 0.25 pH units in response to external ATP in seedling root tips. We validated this pH-Ca2+ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca2+ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca2+ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca2+ signatures act in concert with pH signatures, possibly providing an additional layer of cellular signal transduction to tailor signal specificity
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