Temperature sensing by plants: the primary characteristics of signal perception and calcium response

Cold elicits an immediate rise in the cytosolic free calcium concentration ([Ca2+]c) of plant cells. We have studied the concerted action of the three underlying mechanisms, namely sensing, sensitisation and desensitisation, which become important when plants in the field are subjected to changes in temperature. We applied different regimes of temperature changes with well-defined cooling rates to intact roots of Arabidopsis thaliana expressing the calcium-indicator, aequorin. Our results indicate that temperature sensing is mainly dependent on the cooling rate, dT/dt, whereas the absolute temperature T is of less importance. Arabidopsis roots were found to be sensitive to cooling rates of less than dT/dt = 0.01 degrees C/s. However, at cooling rates below 0.003 degrees C/s (i.e. cooling 10 degrees C in 1 h) there is no detectable [Ca2+]c response at all. At low temperature, the sensitivity of the plant cold-detection system is increased. This in turn produces greater cooling-induced [Ca2+]c elevations. Prolonged or repeated cold treatment attenuates the [Ca2+]c responses to subsequent episodes of cooling.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Release of extracellular purines from plant roots and effect on ion fluxes

Extracellular purine nucleotides appear capable of regulating plant development, defence and stress responses by acting in part as agonists of plasma membrane calcium channels. Factors stimulating ATP release include wounding, osmotic stress and elicitors. Here we show that exogenous abscisic acid and L-glutamate can also cause ATP accumulation around Arabidopsis thaliana roots. Release of ADP from root epidermis would trigger ionotropic receptor-like activity in the plasma membrane, resulting in transient elevation of cytosolic free calcium. Root epidermal protoplasts (expressing aequorin as a cytosolic free calcium reporter) can support an extracellular ADP-induced cytosolic calcium elevation in the presence of an extracellular reductant. This confirms that ADP could elicit calcium-based responses distinct to those of ATP, which have been shown previously to involve production of extracellular reactive oxygen species.     

Mitochondrial calcium in relaxed and tetanized myocardium.

The elemental composition of rat cardiac muscle was determined with electron probe x-ray microanalysis (EPMA) of rapidly frozen papillary muscles and trabeculae incubated with ryanodine (1 microM) in either 1.2 or 10 mM [Ca2+]o-containing solutions, paced at 0.6 Hz or tetanized at 10 Hz. Total mitochondrial calcium increased significantly, by 4.2 mmol/kg dry weight during a 7 s tetanus, only in muscles tetanized in the presence of 10 mM [Ca2+]o when cytoplasmic Ca2+ is 1-4 microM (Backx, P. H., W.-D. Gao, M. D. Azan-Backx, and E. Marban. 1995. The relationship between contractile force and intracellular [Ca2+] in intact rat trabeculae. J. Gen. Physiol. 105:1-19). Comparison of total mitochondrial with free mitochondrial Ca2+ reported in the literature indicates that the total/free ratio is approximately 6000 at physiological or near-physiological levels of total mitochondrial calcium. Increases in free mitochondrial [Ca2+] consistent with regulation of mitochondrial enzymes should be associated with increases in total mitochondrial calcium detectable with EPMA. However, such increases in mitochondrial calcium occur only as the result of prolonged, unphysiological elevations of cytosolic [Ca2+].     

An heuristic hypothesis of chilling injury in plants: a role for calcium as the primary physiological transducer of injury

Abstract. It is suggested that increased levels of free cytosolic calcium ([Ca²⁺]_cyt) may serve as the primary physiological transducer of chilling injury in plants. Numerous similarities between the effects of [Ca²⁺]_cyt-raising treatments on plants and the effects of chilling temperatures on chilling-sensitive (CS) plants are noted. It is proposed that chilling temperatures may lead to increases in [Ca²⁺]_cyt in CS plant cells by reducing the rate at which they exclude Ca²⁺ from their cytosol and that rapid cooling (coldshock) may cause rapid increases in [Ca²⁺]_cyt due to the activation of voltage-dependent cation channels. Chill-induced increases in [Ca²⁺]_cyt in the cells of CS plants may reflect either an inherent inability of such plants to maintain homeostatic levels of Ca²⁺ at low temperatures or a stress-induced reaction which has evolved to enable such cells to cope more effectively with the short-term hardships imposed by cold. Previous proposals concerning the physiological transduction of chilling injury are also discussed. It is argued that there is little evidence to suggest that the immediate effects of low temperatures on CS cells include either decreases in ATP levels, general increases in the passive permeability of membranes, or increased rates of fermentation.     

Cyclosporine augments receptor-mediated cellular Ca2+ fluxes in isolated hepatocytes

The immunosuppressive agent, cyclosporine, has been found to augment receptor-stimulated calcium fluxes in isolated hepatocytes. After treatment of Quin 2-loaded hepatocytes with cyclosporine, both the amplitude and duration of the vasopressin-induced rise in the cytosolic free Ca2+ are increased. These effects are dependent upon the concentration and time of exposure of the cells to cyclosporine. Cyclosporine increases both 45Ca2+ influx across the plasma membrane and the cellular calcium content. The total cellular magnesium, sodium, and potassium contents are not affected by cyclosporine. However, cyclosporine treatment, per se, has no apparent effect on the cytosolic free Ca2+ concentration as assayed by Quin 2 fluorescence. The increase in total cell calcium is associated with progressive increases in the calcium content of the endoplasmic reticular and mitochondrial calcium pools. The vasopressin-induced net efflux of Ca2+ from hepatocytes was 2-fold greater after treatment with 10 micrograms/ml cyclosporine for 10 min, but the lag time prior to the onset of Ca2+ efflux was not affected. These results are interpreted on the basis of cyclosporine having a primary effect on increasing the permeability of the plasma membrane to Ca2+, thereby leading to an increase of the calcium content of the hormone-sensitive intracellular calcium pool.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.     

Conditional and unconditional inhibition of calcium-activated potassium channels by reversible protein phosphorylation

Large conductance, calcium-activated potassium channels (BK(Ca) or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulation of native BK(Ca) channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GH(4)C(1)). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BK(Ca) channel activity approximately 3-fold. Dephosphorylated channels could be activated fully by physiological increases in cytoplasmic calcium or membrane depolarization. In contrast, endogenous protein kinases inhibited BK(Ca) channel activity at two functionally distinct sites. A closely associated, cAMP-dependent protein kinase rapidly reduced channel activity in a conditional manner that could be overcome completely by increasing cytoplasmic free calcium 3-fold or 20 mV further depolarization. Phosphorylation at a pharmacologically distinct site inhibited channel activity unconditionally by reducing availability to approximately half that of maximum at all physiological calcium and voltages. Conditional versus unconditional inhibition of BK(Ca) channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.     

A 42-kilodalton annexin-like protein is associated with plant vacuoles.

A 42-kD, calcium-dependent, membrane-binding protein (VCaB42) was associated with partially purified vacuole membrane. Membrane-dissociation assays indicated that VCaB42 binding to vacuole membranes was selective for calcium over other cations and that 50% of VCaB42 remained membrane bound at 61 +/- 11 nM free calcium. A 13-amino acid sequence obtained from VCaB42 showed 85% similarity with the endonexin fold, a sequence found in the annexin family of proteins that is thought to be essential for calcium and lipid binding. The greatest similarity in amino acid sequence was observed with annexin VIII (VAC-beta). The calcium-binding properties and sequence similarities suggest that VCaB42 is a member of the annexin family of calcium-dependent, membrane-binding proteins. Functional assays for VCaB42 on vacuole membrane transport processes indicated that it did not significantly affect the initial rate of calcium uptake into vacuole membrane vesicles. Because VCaB42 is vacuole localized (likely on the cytosolic surface of the vacuole) and is 50% dissociated within the physiological range of cytosolic free calcium, we hypothesize that this protein is a sensor that monitors cytosolic calcium levels and transmits that information to the vacuole.     

Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.     

Calcium: a fundamental regulator of intracellular membrane fusion?

For many years, it has been known that an increase in cytosolic calcium triggers the fusion of secretory granules and synaptic vesicles with the plasma membrane. However, the role of calcium in the intracellular membrane-fusion reactions that coordinate the secretory and endocytic pathways has been less clear. Initially, there was accumulating evidence to indicate that a focally localized and transient calcium signal is required to trigger even those fusion events formerly classified as 'constitutive'-that is, those that normally occur in the absence of global cytosolic calcium increases. Therefore, calcium seemed to be a required fundamental co-factor underlying all biological membrane-fusion steps, perhaps with a conserved mechanism of action. However, although such unification would be gratifying, new data indicate that several intracellular fusion events do not require calcium after all. In this review, the evidence for calcium requirements and its modes of action in constitutive trafficking are discussed. As a challenging perspective, I suggest that the specific absence of calcium requirements for some transport steps in fact expands the function of calcium in trafficking, because divergent luminal calcium concentrations and requirements for fusion might increase the specificity with which intracellular membrane-fusion partners are determined.     

Ion channels activated by specific Ti or T3 antibodies in plasma membranes of human T cells.

T lymphocytes are activated to proliferate via a surface membrane receptor recognizing the antigen/major histocompatibility complex. This membrane component is comprised of at least five polypeptide subunits, collectively termed the Ti-T3 receptor complex. A transient increase in cytosolic free calcium occurs as an early event in the T-cell activation process and is necessary for induction of the endogenous IL-2 and certain other genes. Monoclonal antibodies specific to epitopes of either the Ti or the T3 components were shown to be effective agonists, also leading to such transient rises in cytosolic free calcium and activating the lymphocytes. Here we show, using micropipette-supported bilayers formed from membranes of the human T-cell line REX, that Ti- or T3-specific antibodies cause opening of ligand gated ion channels. Both types of specific antibodies yielded similar histograms of conductance amplitudes which show a channel with a conductance of 2-3 pS in symmetrical 100 mM CaCl2 solutions. These channels allow the passage of calcium and barium ions and are blocked by lanthanum ions, suggesting that they are specific for calcium. We propose that these channels, by allowing the entry of external calcium, may account for a large fraction of the rise in intracellular calcium observed upon triggering of the Ti-T3 receptor.     

Modelling and experimental analysis of the role of interacting cytosolic and vacuolar pools in shaping low temperature calcium signatures in plant cells

A major challenge to understanding low temperature calcium signatures in plants is defining how these signatures emerge from the interactions of different molecular components that are stored in different subcellular pools of a plant cell. Here we develop an integrative model that incorporates the interactions of Ca2?, H?, K?, Cl? and ATP in both cytosolic and vacuolar pools. Our analysis reveals how these four major ions along with ATP forms a complex network to relate the emergence of calcium signatures to other responses (e.g. pH response). Modelling results are in agreement with experimental observations for both cytosolic free calcium concentration ([Ca2?](c)) and pH. The model is further validated by experimentally measuring the response of [Ca2?](c) to six fluctuating (rather than constant) temperature profiles. We found that modelling results are in reasonable agreement with experimental observations, in particular, if the rate of reducing temperature is relatively high. In addition, we show that both calcium-induced calcium release (CICR) at the vacuolar membrane and transport of ions from the cytosolic pool to the vacuolar membrane play important roles in the interaction between cytosolic and vacuolar pools. In combination they control the amount and timing of calcium release from the vacuolar to cytosolic pool, shaping the specific calcium signature. The methodology and principles developed here establish an integrative view on the role of cytosolic and vacuolar pools in shaping calcium signatures in general, and they are universally applicable to study of the interactions of multiple subcellular pools.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Modulation of membrane potential and ionic currents by the AT1 and AT2 receptors of angiotensin II

Angiotensin II, the principal effector of the renin-angiotensin system, modulates various ionic currents. Its effects on potassium currents, including outward transient potassium current, the inward or outward rectifiers, as well as Ca²⁺-activated potassium currents, is well described. Other ionic currents, such as voltage-dependent calcium currents, cationic or chloride currents, are also altered by the hormone. All these effects provoke changes in membrane potential, such as modulation of action potential firing or resting membrane potential and control intracellular calcium concentration. Summarized here are the results obtained on these membrane electrical properties using electrophysiological recordings.     

Electrophysiological evidence for a role for calcium in temperature sensing by roots of cucumber seedlings

Abstract. Rapid-cooling pulses to non-stressful temperatures cause strong, transient depolarizations in cortical cells of cucumber roots. The amplitudes of these electrical responses are graded according to the rate and amplitude of the cooling pulse. Such graded potentials are typical of sensory processes and indicate that plants possess the ability to sense temperature change. La³⁺, a blocker of Ca²⁺ channels, and ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-acetic acid (EGTA), a Ca²⁺ chelator, inhibit the electrical responses elicited by rapid-cooling pulses. High external [Ca²⁺] enhances them. These results indicate the involvement of a plasma membrane-associated Ca²⁺ channel in the process of temperature sensing by plants. Calmodulin antagonists prolong the repolarization phase of the electrical responses, suggesting a role for calmodulin in the recovery from stimulation.     

Oxidative Signals in Tobacco Increase Cytosolic Calcium

Tobacco (Nicotiana plumbaginifolia) seedlings genetically transformed to express apoaequorin were incubated in h-coelenterazine to reconstitute the calcium-sensitive luminescent protein aequorin. Treatment of these seedlings with hydrogen peroxide resulted in a transient burst of calcium-dependent luminescence lasting several minutes. Even though the hydrogen peroxide stimulus was persistent, the change in cytosolic free calcium concentration ([Ca2+]cyt) was transient, suggesting the presence of a refractory period. When seedlings were pretreated with hydrogen peroxide, there was no increase in [Ca2+]cyt upon a second application, which confirmed the refractory character of the response. Only when the two treatments were separated by 4 to 8 hr was full responsiveness recovered. However, treatment with hydrogen peroxide did not inhibit mobilization of [Ca2+]cyt induced by either cold shock or touching, suggesting that these three signals mobilize different pools of intracellular calcium. To examine whether [Ca2+]cyt is regulated by the redox state of the cytoplasm, we pretreated seedlings with buthionine sulfoximine (to modify cellular glutathione levels) and inhibitors of ascorbate peroxidase. These inhibitors modify the hydrogen peroxide-induced transients in [Ca2+]cyt, which is consistent with their effects on the cellular prooxidant/antioxidant ratio. Treatment with hydrogen peroxide that elicited [Ca2+]cyt increases also brought about a reduction in superoxide dismutase enzyme activity. This reduction could be reversed by treatment with the calcium channel blocker lanthanum. This indicates that there is a role for calcium in plant responses to oxidative stress.     

Cobalt activates potassium conductance in the plasma membrane of cultured renal epithelioid (MDCK)-cells

Cobalt has been shown to stimulate sodium transport across the distal nephron of the newt kidney. The mechanism of this action remained elusive. The present study has been performed to test for effects of cobalt on electrical properties of cultured subconfluent kidney (MDCK)-cells: cobalt (10 microM) leads to a rapid, sustained and reversible hyperpolarization of the cell membrane, paralleled by an increase of the potassium selectivity and a decrease of the resistance. Thus, cobalt increases the potassium conductance of the cell membrane. The half-maximal effect is elicited by approx. 1 microM. At extracellular calcium concentration reduced to less than 0.1 microM, cobalt (10 microM) leads to a transient hyperpolarization, which can be elicited only once. Thus, cobalt enhances the potassium conductance in a calcium dependent way. At higher concentrations (100 microM) cobalt hyperpolarizes the cell membrane only transiently even in the presence of extracellular calcium. Furthermore 100 microM cobalt interferes with ATP-induced hyperpolarization, which is known to result from calcium mediated activation of K+ channels. Thus, 100 microM cobalt may inhibit ATP-stimulated calcium entry into the cell.     

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering by cytoplasmic calcium binding proteins. Numerical integration of the model allows us to study the fluctuations in the cytosolic calcium concentration, the ER membrane potential, and the concentration of free calcium binding sites on a calcium binding protein. The model demonstrates the physiological features necessary for calcium oscillations and suggests that the level of calcium flux into the cytosol controls the frequency and amplitude of oscillations. The model also suggests that the level of buffering affects the frequency and amplitude of the oscillations. The model is supported by experiments indirectly measuring cytosolic calcium by calcium-induced chloride currents in Xenopus oocytes as well as cytosolic calcium oscillations observed in other preparations.     

ATP can stimulate exocytosis in rat brown adipocytes without apparent increases in cytosolic Ca2+ or G protein activation

Extracellular ATP activates large increases in cell surface area and membrane turnover in rat brown adipocytes (Pappone, P. A., and Lee, S. C. 1996. J. Gen. Physiol. 108:393-404). We used whole-cell patch clamp membrane capacitance measurements of membrane surface area concurrently with fura-2 ratio imaging of intracellular calcium to test whether these purinergic membrane responses are triggered by cytosolic calcium increases or G protein activation. Increasing cytosolic calcium with adrenergic stimulation, calcium ionophore, or calcium-containing pipette solutions did not cause exocytosis. Extracellular ATP increased membrane capacitance in the absence of extracellular calcium with internal calcium strongly buffered to near resting levels. Purinergic stimulation still activated exocytosis and endocytosis in the complete absence of intracellular and extracellular free calcium, but endocytosis predominated. Modulators of G protein function neither triggered nor inhibited the initial ATP-elicited capacitance changes, but GTPgammaS or cytosolic nucleotide depletion did reduce the cells' capacity to mount multiple purinergic responses. These results suggest that calcium modulates purinergically-stimulated membrane trafficking in brown adipocytes, but that ATP responses are initiated by some other signal that remains to be identified.