Patrice Thuleau

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.

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.

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.

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.

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.

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium dependent programmed cell death (PCD) in tobacco BY-2 cells. In addition, we have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction.

Signal transduction and calcium channels in higher plants

Abstract An improved understanding of calcium signal transduction in higher plants has been reached by using various modern approaches, such as fluorescent calcium imaging techniques, patch-clamp studies, biochemical studies of plant membranes and the construction of plants transformed with the apoaequorin gene. Some of the most recent promising advances in this field are discussed in this review.

Putative L-Type Calcium Channels in Plants: Biochemical Properties and Subcellular Localisation

It is generally recognised that many essential cellular responses are regulated by the cytoplasmic free calcium ion concentration (Poovaiah and Reddy, 1987). Changes in [Ca] levels are detected by proteins and enzymes whose activities are modified in response to altered calcium levels (Hepler and Wayne, 1985: Ranjeva and Boudet, 1987); this results in the control of metabolism, gene expression and integrated functions such as exocytosis (Braam and Davis, 1990; Steer, 1988). For these reasons, calcium is considered as an important second messenger in eukaryotic organisms.

Control of free calcium in plant cell nuclei : Cell signalling

Free calcium ions stimulate a huge variety of processes inside the cell, eliciting specific responses that depend on their spatio-temporal concentrations. Here we investigate how these changes in calcium concentration are triggered in the cytosol and nucleus of plant cells, and find that they are independently controlled in the two compartments. Our results indicate that in plants some processes in the nucleus may be executed in response to an autonomously regulated nuclear calcium signal, although it is not clear whether this happens in animal cells as well.