Papers by Christian Mazars
Canadian Journal of Botany, 1989
ABSTRACT Small concentrations of a high molecular weight phytotoxic glycoprotein obtained from cu... more ABSTRACT Small concentrations of a high molecular weight phytotoxic glycoprotein obtained from culture filtrates of Rhynchosporium secalis (Oud.) Davis induced chlorosis and necrosis in barley; these symptoms were similar to those observed during the final stages of disease caused by this pathogen in naturally infected barley leaves. Histochemical studies indicated that cells of leaf tissues treated with the glycoprotein became disorganized. We observed changes such as the collapse of epidermal anticlinal walls, tissue necrosis, and plugging of xylem vessels. Histochemical and immunofluorescent techniques that localized the glycoprotein toxin indicated that, at least in part, the plugging results from a plant response stimulated by the toxic glycoprotein.
Signaling and Communication in Plants, 2011
Calcium variations occurring in the nucleus and in other calcium-active compartments of the plant... more Calcium variations occurring in the nucleus and in other calcium-active compartments of the plant cell are contributing to encode information of specificity used by the cell to mount an appropriate response to environmental cues. This chapter deals with calcium signaling in the nucleus and reports on the current knowledge on calcium signals monitored in plant cell nuclei in response to
Frontiers in Plant Science, 2014
Calcium (Ca 2+ ) is a second messenger involved in many plant signaling processes. Biotic and abi... more Calcium (Ca 2+ ) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca 2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca 2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca 2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca 2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca 2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca 2+ -dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca 2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca 2+ dynamics in intact plants. We describe a simple method to image Ca 2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca 2+ signals. It is shown that Ca 2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.
Biochimie, 2015
MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the... more MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the regulation of different cellular processes. However in plants, little is known about the regulation of these Dual Specific Phosphatases (DSPs) by Ca(2+) and calmodulin (CaM). Here, we showed that the wheat MKP (TMKP1) harboring a calmodulin (CaM) binding domain, binds to CaM in a Ca(2+)-dependent manner. In addition, TMKP1 exhibited a phosphatase activity in vitro that is specifically enhanced by Mn(2+) and to a lesser extent by Mg(2+), but without any synergistic effect between the two bivalent cations. Most interestingly, CaM/Ca(2+) complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent manner. However, in the presence of Mn(2+) this activity is enhanced by CaM/Ca(2+) complex. These dual regulatory effects seem to be mediated via interaction of CaM/Ca(2+) to the CaM binding domain in the C-terminal part of TMKP1. Such effects were not reported so far, and raise a possible role for CaM and Mn(2+) in the regulation of plant MKPs during cellular response to external signals.
Plant Signaling & Behavior, 2014
Growing plants in space for using them in bioregenerative life support systems during long-term h... more Growing plants in space for using them in bioregenerative life support systems during long-term human spaceflights needs improvement of our knowledge in how plants can adapt to space growth conditions. In a previous study performed on board the International Space Station (GENARA A experiment STS-132) we evaluate the global changes that microgravity can exert on the membrane proteome of Arabidopsis seedlings. Here we report additional data from this space experiment, taking advantage of the availability in the EMCS of a centrifuge to evaluate the effects of cues other than microgravity on the relative distribution of membrane proteins. Among the 1484 membrane proteins quantified, 227 proteins displayed no abundance differences between µ g and 1 g in space, while their abundances significantly differed between 1 g in space and 1 g on ground. A majority of these proteins (176) were over-represented in space samples and mainly belong to families corresponding to protein synthesis, degradation, transport, lipid metabolism, or ribosomal proteins. In the remaining set of 51 proteins that were under-represented in membranes, aquaporins and chloroplastic proteins are majority. These sets of proteins clearly appear as indicators of plant physiological processes affected in space by stressful factors others than microgravity.
PLoS ONE, 2014
The ''GENARA A'' experiment was designed to monitor global changes in the proteome of membranes o... more The ''GENARA A'' experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in mg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in mg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.
The Plant Journal, 1998
Depolarization-activated plasma membrane calcium channels have been suggested to play prominent r... more Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch-clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein-protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch-clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life threetimes longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels 603 in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.
The Plant Journal, 2009
Calcium (Ca 2+ ), as a second messenger, is crucial for signal transduction processes during many... more Calcium (Ca 2+ ), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca 2+ ] elevations are early events in the interaction between the plant growth-promoting fungus Piriformospora indica and Arabidopsis thaliana. A cell wall extract (CWE) from the fungus promotes the growth of wild-type seedlings but not of seedlings from P. indica-insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca 2+ signalling-related functions. The CWE induces a transient cytosolic Ca 2+ ([Ca 2+ ] cyt ) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum) plants, as well as in BY-2 suspension cultures expressing the Ca 2+ bioluminescent indicator aequorin. Nuclear Ca 2+ transients were also observed in tobacco BY-2 cells. The Ca 2+ response was more pronounced in roots than in shoots and involved Ca 2+ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca 2+ response by staurosporine and the refractory nature of the Ca 2+ elevation suggest that a receptor may be involved. The CWE does not stimulate H 2 O 2 production and the activation of defence gene expression, although it led to phosphorylation of mitogen-activated protein kinases (MAPKs) in a Ca 2+ -dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica. Thus, Ca 2+ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.
THE PLANT CELL ONLINE, 2002
Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin... more Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca 2 ؉ ] cyt ) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration.
PLANT PHYSIOLOGY, 2007
Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresse... more Proline (Pro) accumulation occurs in various plant organisms in response to environmental stresses. To identify the signaling components involved in the regulation of Pro metabolism upon water stress in Arabidopsis (Arabidopsis thaliana), a pharmacological approach was developed. The role of phosphoinositide-specific phospholipases C (PLCs) in Pro accumulation was assessed by the use of the aminosteroid U73122, a commonly employed specific inhibitor of receptor-mediated PLCs. We found that U73122 reduced pyrroline-5-carboxylate synthetase transcript and protein as well as Pro levels in salt-treated seedlings. Inhibition of PLC activity by U73122 was quantified by measuring the decrease of inositol 1,4,5-trisphosphate (InsP 3 ) levels. Moreover, the utilization of diacylglycerol kinase and InsP 3 -gated calcium release receptor inhibitors suggested that InsP 3 or its derivatives are essential for Pro accumulation upon salt stress, involving calcium as a second messenger in ionic stress signaling. This observation was further supported by a partial restoration of Pro accumulation in salt-and U73122-treated seedlings after addition of extracellular calcium, or when calcium homeostasis was perturbed by cyclopiazonic acid, a blocker of plant type IIA calcium pumps. Taken together, our data indicate that PLC-based signaling is a committed step in Pro biosynthesis upon salinity but not in the case of mannitol stress. Calcium acts as a molecular switch to trigger downstream signaling events. These results also demonstrated the specific involvement of lipid signaling pathway to discriminate between ionic and nonionic stresses.
New Phytologist, 2009
In plant cells, calcium-based signaling pathways are involved in a large array of biological proc... more In plant cells, calcium-based signaling pathways are involved in a large array of biological processes, including cell division, polarity, growth, development and adaptation to changing biotic and abiotic environmental conditions. Free calcium changes are known to proceed in a nonstereotypical manner and produce a specific signature, which mirrors the nature, strength and frequency of a stimulus. The temporal aspects of calcium signatures are well documented, but their vectorial aspects also have a profound influence on biological output. Here, we will focus on the regulation of calcium homeostasis in the nucleus. We will discuss data and present hypotheses suggesting that, while interacting with other organelles, the nucleus has the potential to generate and regulate calcium signals on its own.
Molecular Plant, 2010
Calcium and Reactive Oxygen Species (ROS) are acknowledged as crucial second messengers involved ... more Calcium and Reactive Oxygen Species (ROS) are acknowledged as crucial second messengers involved in the response to various biotic and abiotic stresses. However, it is still not clear how these two compounds can play a role in different signaling pathways leading the plant to a variety of processes such as root development or defense against pathogens. Recently, it has been shown that the concept of calcium and ROS signatures, initially discovered in the cytoplasm, can also be extended to the nucleus of plant cells. In addition, it has been clearly proved that both ROS and calcium signals are intimately interconnected. How this cross-talk can finally modulate the translocation and/or the activity of nuclear proteins leading to the control of specific genes expression is the main focus of this review. We will especially focus on how calcium and ROS interact at the molecular level to modify their targets.
Journal of Experimental Botany, 2010
The laterals of both shoots and roots often maintain a particular angle with respect to the gravi... more The laterals of both shoots and roots often maintain a particular angle with respect to the gravity vector, and this angle can change during organ development and in response to environmental stimuli. However, the cellular and molecular mechanisms of the lateral organ gravitropic response are still poorly understood. Here it is demonstrated that the young siliques of Arabidopsis thalinana plants subjected to 3-D clinostat rotation exhibited automorphogenesis with increased growth angles between pedicels and the main stem. In addition, the 3-D clinostat rotation treatment significantly influenced the development of vascular bundles in the pedicel and caused an enlargement of gap cells at the branch point site together with a decrease in KNAT1 expression. Comparisons performed between normal and empty siliques revealed that only the pedicels of siliques with normally developing seeds could change their growth angle under the 3-D clinostat rotational condition, while the pedicels of the empty siliques lost the ability to respond to the altered gravity environment. These results indicate that the response of siliques to altered gravity depends on the normal development of seeds, and may be mediated by vascular bundle cells in the pedicel and gap cells at branch point sites.
Journal of Experimental Botany, 2009
Calcium-mediated signalling is ubiquitous in both animals and plants. Changes in cytoplasmic free... more Calcium-mediated signalling is ubiquitous in both animals and plants. Changes in cytoplasmic free Ca(2+) concentration couple diverse arrays of stimuli to their specific responses, the specificity of the stimulus being determined by integrated actions between multiple Ca(2+) mobilization pathways. In this work, a pharmacological approach is reported, aimed at deciphering the role of calcium as a second messenger in the transduction pathway leading to the inhibitory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), in regulating monoterpene indole alkaloid (MIA) biosynthesis in Catharanthus roseus cells. It is demonstrated here that auxin-dependent MIA biosynthesis is differentially regulated by two distinct calcium release components from internal stores in C. roseus showing pharmacological profiles similar to those displayed by animal RyR and IP3 channels. MIA biosynthesis is stimulated by caffeine (Ca(2+)-release activator through RyR channels) and by heparin and TMB8 (Ca(2+)-release inhibitors of IP3 channels) whereas MIA biosynthesis is inhibited by mastoparan (Ca(2+)-release activator of IP3 channels) and by ruthenium red and DHBP (Ca(2+)-release inhibitors of RyR channels). Furthermore, calcium, as 2,4-D, acts on MIA biosynthesis by regulating the monoterpene moiety of the MIA biosynthesis pathway since calcium channel modulators preferentially modulate g10h expression, the gene encoding the enzyme of the secoiridoid monoterpene pathway, that is the major target of 2,4-D action. In addition, the simultaneous use of caffeine (an activator of RyR channel in animals) and TMB8 (an inhibitor of the IP3 channel) in 2,4-D treated cells triggers a synergistic effect on MIA accumulation. This finding suggests an opposite and co-ordinated action of multiple Ca(2+)-release pathways in 2,4-D signal transduction, adding a new level of complexity to calcium signalling in plants and questioning the existence of RyR and IP3 channels in plants.
FEBS Letters, 1996
Plasma membrane-bound voltage-dependent calcium channels may couple the perception of an initial ... more Plasma membrane-bound voltage-dependent calcium channels may couple the perception of an initial stimulus to a regulated pathway for calcium influx. The activities of these channels have been shown to be very low and highly unstable but may be recruited by large-predepolarizing pulses, according to a process referred to as recruitment. By combining pharmacological and electrophysiological approaches, we demonstrate in the present paper that the cytoskeleton plays an important role in the regulation of the activity and stability of voltage-dependent calcium channels during whole-cell patch-clamp experiments on carrot protoplasts. Whereas drugs affecting the organization of the microfilament network have no measurable effect, the manipulation of the microtubule network elicits important changes. Thus, the addition of colchicine or oryzalin, which are known to disrupt microtubule organization, leads to a 6-10-fold increase in calcium channel activities and half-life. In contrast, stabilization of the microtubules by taxol has no effect on any of these parameters. The data obtained suggest that interactions of microtubules and voltage-dependent calcium channels by either direct or indirect mechanisms inhibit channel activities and decrease their half-life. In contrast, the disruption of the network overcomes such an inhibitory effect and allows the activation of calcium channels. It is speculated that under normal physiological conditions these protein-protein interactions may work in a reversible manner and contribute to signal transduction in higher plants.
FEBS Letters, 2006
The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous gl... more The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.
Cell Calcium, 1997
Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium ... more Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.
Cell Calcium, 2005
We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitor... more We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca 2+ , as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca 2+ ] nuc ) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the -1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca 2+ ] nuc elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca 2+ ] nuc elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca 2+ ] nuc rise depends on free cytosolic calcium, IP 3 , and active oxygen species (AOS) but is independent of nitric oxide.
Cell Calcium, 2001
Plant cell suspension cultures respond to osmotic changes by alterations in levels of free cellul... more Plant cell suspension cultures respond to osmotic changes by alterations in levels of free cellular calcium. Using the aequorin recombinant method, we have measured the spatial and temporal characteristics of calcium signatures in the nucleus and the cytosol of BY-2 tobacco suspension cells challenged with hypo- or hyper-osmotic shock. We show here that the nuclear compartment contributes together with the cytosol to produce calcium signal patterns that discriminate hypo- from hyper-osmotic treatments, i.e. turgor from tension. We also demonstrate that calcium responses in the nucleus and the cytosol are differentially modulated by the strength and the nature of hyper-osmotic treatments. We conclude that qualitative and quantitative changes in the parameters of an external stimulus such as osmotic changes are converted into calcium signatures, distinctive in their temporal and subcellular characteristics, involving both the nucleus and the cytosol. Our results illustrate the versatility of calcium signaling in plant cells. In addition to the physiological 'address' of the cell, the compartmentation of the calcium signal is probably an important parameter in encoding response specificity.
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Papers by Christian Mazars