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P Thuleau

Publications and source records attributed to P Thuleau.

12 recordsLinked to original sources
PubMed2001

The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells.

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.

Calcium Channel Blockers↗
PubMed2001

[Basis for the calcium specificity in the plant response to extracellular stimulation].

The Ca2+ cation is fully recognized as an important intracellular second messenger coupling a wide range of extracellular stimuli to characteristic responses in plant cells. Such a pleiotropic effect raises questions regarding the mechanisms by which the signalling pathways, all of then involving an increase in intracellular calcium concentration, can be specific to a given stimulus. Here, we present recent results which shed light into different concepts which may explain the response specificity in signalling processes, such as "the cross-talk between signalling pathways", "the Ca2+ signatures" and "the compartmentation of Ca(2+)-signalling".

Calcium↗
PubMed1998

Recent advances in the regulation of plant calcium channels: evidence for regulation by G-proteins, the cytoskeleton and second messengers.

Important aspects of the regulatory properties of plant calcium channels have been discovered during the past few years. These include the control of plasma membrane-bound channels by regulatory proteins and the characterization of a plethora of intracellular calcium release channels. Deciphering the mechanisms of regulation of different Ca2+ channels and the probable co-operation of their activities in response to various stimuli is leading to a better understanding of Ca2+-signalling processes in higher plants.

Abscisic Acid↗
PubMed1998

Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules.

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 three-times 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 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.

Arabidopsis↗
PubMed1997

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

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.

Actin Cytoskeleton↗
PubMed1996

Activation of plasma membrane voltage-dependent calcium-permeable channels by disruption of microtubules in carrot cells.

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.

Calcium Channels↗
PubMed1994

Recruitment of plasma membrane voltage-dependent calcium-permeable channels in carrot cells.

Numerous biological assays and pharmacological studies have led to the suggestion that depolarization-activated plasma membrane Ca2+ channels play prominent roles in signal perception and transduction processes during growth and development of higher plants. The recent application of patch-clamp techniques to isolated carrot protoplasts has led to direct voltage-clamp evidence for the existence of Ca2+ channels activated by physiological depolarizations in the plasma membrane of higher plant cells. However, these voltage-dependent Ca2+ channels were not stable and their activities decreased following the establishment of whole-cell recordings. We show here that large pre-depolarizing pulses positive to 0 mV induced not only the recovery of Ca2+ channel activities, but also the activation of initially quiescent voltage-dependent Ca2+ channels in the plasma membrane (recruitment). This recruitment was dependent on the intensity and duration of membrane depolarizations, i.e. the higher and longer the pre-depolarization, the greater the recruitment. Pre-depolarizing pulses to +118 mV during 30 s increased the initial calcium currents 5- to 10-fold. The recruited channels were permeable to Ba2+ and Sr2+ ions. The data suggested that voltage-dependent Ca(2+)-permeable channels are regulated by biological mechanisms which might be induced by large pre-depolarizations of the plasma membrane. In addition, this study provides evidence for the existence in the plasma membrane of higher plant cells of a large number of voltage-dependent Ca2+ channels of which a major part are inactive and quiescent. It is suggested that quiescent Ca2+ channels can be rapidly recruited for Ca(2+)-dependent signal transduction.

Calcium↗
PubMed1994

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Calcium Channels↗
PubMed1993

Solubilized proteins from carrot (Daucus carota L.) membranes bind calcium channel blockers and form calcium-permeable ion channels.

Calcium channels have been suggested to play a major role in the initiation of a large number of signal transduction processes in higher plant cells. However, molecular components of higher plant Ca2+ channels remain unidentified to date. Calcium channel blockers of the phenylalkylamine family and bepridil specifically inhibit Ca2+ influx into carrot (Daucus carota L.) cells. By using a phenylalkylamine azido derivative, a 75-kDa carrot membrane protein has been previously identified. Here we have partially purified this Ca2+ channel blocker-binding protein by lectin-affinity and ion-exchange chromatographies. The protein fraction containing the 75-kDa binding protein was incorporated into giant liposomes. Single-channel patch-clamp studies on these proteoliposomes showed the presence of Ca2+-permeable channel currents. These Ca2+-permeable channels were not stable. Recordings after durations of 2-10 min showed the appearance of nonselective ion channels with a permeability to calcium and chloride ions. These nonselective Ca2+-permeable ion channels, in contrast, were stable and were recorded for extended durations. The addition of the Ca2+ channel-blocker bepridil (10 M) led to the inhibition of these nonselective Ca2+-permeable channels by reducing the probability of channel opening. These results suggest that the 75-kDa Ca2+ channel blocker-binding protein from carrot cells plays a role in channel sensitivity to Ca2+ channel inhibitors and may constitute one of the components of Ca2+ channels in higher plants.

Journal Article↗
PubMed1990

A 75-kDa polypeptide, located primarily at the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888.

Calcium channel blockers of the phenylalkylamine family bind specifically to membranes and inhibit calcium uptake in carrot protoplast. LU 49888, an azido derivative of phenylalkylamine, behaves as its unmodified homolog in terms of affinity and specificity and therefore allows us to probe the receptor by photoaffinity labeling. Upon UV irradiation, a 75-kDa peptide was specifically labeled. Incubation of microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent, led to the solubilization of the LU 49888-binding protein. Electrophoretic analysis under denaturing conditions and gel filtration of the solubilized "receptor-ligand" complex show a 75-kDa peptide mainly located at the plasma membrane. Consequently the LU 49888-binding protein in plants differs significantly from its animal counterpart by its size and may be a primary target for external signal molecules.

Journal Article↗
PubMed1988

Binding of the phytotoxin zinniol stimulates the entry of calcium into plant protoplasts.

Zinniol [1,2-bis(hydroxymethyl)-3-methoxy-4-methyl-5-(3-methyl-2-butenyloxy)benzene], a toxin produced by fungi of the Alternaria group, causes symptoms in plants that resemble those induced by the fungi. The phytotoxin binds to carrot protoplasts and isolated membranes in a saturable and reversible manner. Receptor occupancy stimulates entry of calcium into protoplasts. Zinniol can partially reverse the effects and binding of the calcium-channel blockers desmethoxyverapamil and bepridil. Selected cell lines that are insensitive to zinniol lose part of their binding capacity and sensitivity to the action of the agonist-like compound but are still able to bind calcium-channel blockers. We conclude that zinniol acts on calcium entry but that the targets of the toxin and of calcium-channel blockers are dissimilar, suggesting the occurrence of sites affected both by zinniol and by channel blockers and of sites affected only by zinniol.

Journal Article↗