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Calcium imaging

Contacts : mail to Christian MAZARS, mail to Patrice THULEAU

Generally, plant cells respond to different stimuli by a change in calcium levels in the cytosol considered for a long time as the only active cell compartment in terms of calcium signaling. Recent data from the literature indicate that other cellular compartments like mitochondria, chloroplasts or nucleus are also involved in the generation of a calcium change in response to specific stimuli (Xiong et al. 2006). Compartmentalization of the calcium signal and other parameters such as duration, intensity, origin, form and frequency are used to describe the calcium variation. These parameters led to the emergence of the concept of calcium signature that assigns to a given stimulus a specific calcium variation (McAinsh & Pittman 2009). The interest of the team was particularly focused from 2000’s until recently on the nuclear compartment that supports the expression of genes required for the implementation of the adaptive response. To address this research theme, the team has developed tools to monitor real-time changes in calcium concentration in both the cytosol and the nucleus. It consists in the natural bioluminescent calcium probe aequorin, extracted from the jellyfish, whose functioning is shown schematically below :

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Figure 1.
Principe de fonctionnement de la sonde calcique aequorine

The apo-aequorin is able in the presence of coelenterazine (chromophore) to bind three calcium ions. This will cause a conformational change of the protein leading to an intramolecular oxidation and to the emission of blue light (468nm) proportional to the concentration of calcium and detectable by the photomultiplier of a luminometer or an intensified camera.

Using this approach the group could highlight :

  • the active contribution of the nucleus to the cell calcium signature
  • the nucleus autonomy in terms of perception of external signals and calcium signaling
  • the possibility that the nucleus may control cellular responses involving calcium, independently or in concert with the cytosol

Current research is now focusing on setting up new tools for real-time imaging of calcium changes in the whole plant or tissues using bioluminescence.

Within the frame of collaborative projects, using aequorin or its derivatives as calcium probes, the aims are to optimize detection systems to visualize the calcium changes in response to various stimuli in a whole plant, organ or tissue with the ultimate goal to visualize calcium changes at a single cell level. This purpose is supported by the fact that aequorin is one of the calcium probe having the highest dynamic range (Alonso et al.2011). As an example, shown in the following video are nuclear calcium peaks in roots of Arabidopsis plantlets constitutively expressing nuclear aequorin and elicited with 500µg/mL chitosan.

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Nuclear calcium in Arabidopsis roots

In this context, Xiong et al (2014) developed a more sensitive calcium probe based on Bioluminescence Resonance Energy Transfer (BRET) those general principle is given below. This new probe has a ratio signal /noise which is 3-5 times higher than aequorin .

As an illustration of the use of this probe is shown in the video the light emission from a 4 week-old G5A-expressing Arabidopsis plant upon a high salt stimulus applied to the roots . The video clearly shows that calcium waves propagates with different speeds according the plant organs (Xiong et al. 2014 ).

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Cytosolic calcium waves in Arabidopsis leaves

Collaborations :

Barker D. , De Carvalho-Niebel F. :

Le Ru A. :

Mithöfer A. :

Xiong T.C. :

GDR calcium :

Related References (external) :

McAinsh MR, Pittman JK. (2009) Shaping the calcium signature. New Phytol. 181(2):275-94.

Alonso MT and Garcia-Sancho J. (2011) Nuclear Ca2+Signalling. Cell Calcium 49:280-289

Related References (group) :

Xiong TC, Ronzier E, Sanchez F, Corratgé-Faillie C, Mazars C, Thibaud JB.(2014). Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter. Front Plant Sci.5:43.

Thuleau P, Brière C, Mazars C.(2012). Recent advances in plant cell nuclear signaling. Mol Plant. 5:968-70.

Ranty B, Cotelle V, Galaud JP, Mazars C.(2012) Nuclear calcium signaling and its involvement in transcriptional regulation in plants. Adv Exp Med Biol. 740:1123-43

Amelot N, Carrouche A, Danoun S, Bourque S, Haiech J, Pugin A, Ranjeva R, Grima-Pettenati J, Mazars C, Briere C.(2011). Cryptogein, a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium-dependent manner. Plant Cell Environ. 1:149-61.

Mazars C, Brière C, Bourque S, Thuleau P.(2011). Nuclear calcium signaling : an emerging topic in plants. Biochimie. 12:2068-74

Lachaud C, Da Silva D, Cotelle V, Thuleau P, Xiong TC, Jauneau A, Brière C, Graziana A, Bellec Y, Faure JD, Ranjeva R, Mazars C. (2010).Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells. Cell Calcium. 1:92-100.

Mazars C, Thuleau P, Lamotte O, Bourque S.(2010). Cross-talk between ROS and calcium in regulation of nuclear activities. Mol Plant. 4:706-18.

Dagher R, Brière C, Fève M, Zeniou M, Pigault C, Mazars C, Chneiweiss H, Ranjeva R, Kilhoffer MC, Haiech J.(2009). Calcium fingerprints induced by calmodulin interactors in eukaryotic cells. Biochim Biophys Acta. 6:1068-77.

Mazars C, Bourque S, Mithöfer A, Pugin A, Ranjeva R. (2009) Calcium homeostasis in plant cell nuclei. New Phytol. 2:261-74.

Mithöfer A, Mazars C, Maffei ME.(2009) Probing Spatio-temporal Intracellular Calcium Variations in Plants. Methods Mol Biol 479:79-92.

Poutrain P, Mazars C, Thiersault M, Rideau M, Pichon O.(2009). Two distinct intracellular Ca2+-release components act in opposite ways in the regulation of the auxin-dependent MIA biosynthesis in Catharanthus roseus cells. J Exp Bot. 4:1387-98

Vadassery J, Ranf S, Drzewiecki C, Mithöfer A, Mazars C, Scheel D, Lee J, Oelmüller R. (2009). A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots. Plant J. 2:193-206.

Xiong TC, Coursol S, Grat S, Ranjeva R, Mazars C.(2008) Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco. Cell Calcium. 2008 1:29-37.

Parre E, Ghars MA, Leprince AS, Thiery L, Lefebvre D, Bordenave M, Richard L, Mazars C, Abdelly C, Savouré A.(2007). Calcium signaling via phospholipase C is essential for proline accumulation upon ionic but not nonionic hyperosmotic stresses in Arabidopsis. Plant Physiol. 144 (1):503-12.

Walter A, Mazars C, Maitrejean M, Hopke J, Ranjeva R, Boland W, Mithöfer A.(2007) Structural requirements of jasmonates and synthetic analogues as inducers of Ca2+ signals in the nucleus and the cytosol of plant cells. Angew Chem Int Ed Engl. 25:4783-5

Xiong TC, Bourque S, Lecourieux D, Amelot N, Grat S, Brière C, Mazars C, Pugin A, Ranjeva R. (2006) Calcium signaling in plant cell organelles delimited by a double membrane. Biochim Biophys Acta. 1763(11):1209-15 Brière C, Xiong TC, Mazars C, Ranjeva R.(2006). Autonomous regulation of free Ca2+ concentrations in isolated plant cell nuclei : a mathematical analysis. Cell Calcium. 2006 39(4):293-303

Xiong TC, Jauneau A, Ranjeva R, Mazars C.(2004). Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling. Plant J. 40(1):12-21

Mithöfer A, Mazars C.(2002). Aequorin-based measurements of intracellular Ca2+-signatures in plant cells. Biol Proced Online. 4:105-118

Lecourieux D, Mazars C, Pauly N, Ranjeva R, Pugin A. (2002). Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells. Plant Cell. 2002 14(10):2627-41

Pauly N, Knight MR, Thuleau P, Graziana A, Muto S, Ranjeva R, Mazars C.(2001). The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells. Cell Calcium. 30(6):413-21.

Pauly N, Knight MR, Thuleau P, van der Luit AH, Moreau M, Trewavas AJ, Ranjeva R, Mazars C. Control of free calcium in plant cell nuclei.(2000) Nature. 405(6788):754-5.