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.     

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.     

Action of aluminum, novel TPC1-type channel inhibitor, against salicylate-induced and cold-shock-induced calcium influx in tobacco BY-2 cells

Previously, effect of Al ions on calcium signaling was assessed in tobacco cells expressing a Ca2+-monitoring luminescent protein, aequorin and a newly isolated putative plant Ca2+ channel protein from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). TPC1 channels were shown to be the only channel known to be sensitive to Al and they are responsive to reactive oxygen species and cryptogein, a fungal elicitor protein. Thus, involvement of TPC1 channels in calcium signaling leading to development of plant defense mechanism has been suggested. Then, the use of Al as a specific inhibitor of TPC1-type plant calcium channels has been proposed. Here, using transgenic tobacco BY-2 cells expressing aequorin, we report on the evidence in support of the involvement of Al-sensitive signaling pathway requiring TPC1-type channel-dependent Ca2+ influx in response to salicylic acid, a key plant defense-inducing agent, but not to an elicitor prepared from the cell wall of rice blast disease fungus Magnaporthe grisea. In addition, involvement of Al-sensitive Ca2+ channels in response to cold shock was also tested. The data suggested that the elicitor used here induces the Ca2+ influx via Al-insensitive path, while salicylic acid and cold-shock-stimulate the influx of Ca2+ via Al-sensitive mechanism.     

Calcium measurement in living filamentous fungi expressing codon-optimized aequorin

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.     

重组水母发光蛋白及其在植物钙离子信号检测中的应用

重组水母发光蛋白作为检测植物细胞钙信号的手段是近十几年发展起来的新方法,该文介绍了重组水母发光蛋白作为Ca2+检测探针的发展过程、测钙原理、Ca2+浓度检测方法、Ca2+浓度换算方法、优点与不足、及在植物细胞钙离子信号检测中的研究进展。并利用国外实验室提供的方法在国内首次得出冷激条件下植物细胞内细胞质中([Ca2+]cyt)和液泡膜附近([Ca2+]md)钙离子浓度动力学变化曲线。     

Bounds on the estimation of calcium by aequorin luminescence in the presence of inhomogeneity.

The photoprotein aequorin, which emits light as a nonlinear function of calcium concentration, is often used to measure intracellular calcium. In the presence of inhomogeneities or fluctuations of calcium concentration, the nonlinearity results in discrepancies between mean calcium concentration estimated from average aequorin light and the true mean. It is usually assumed that the error is an overestimation, but in the presence of large calcium fluctuations, errors of either direction are possible. Here we show that for aequorin to overestimate the mean calcium, the point in the calcium-light plane representing the true mean calcium and measured mean aequorin light must lie in the convex envelope of that segment of the aequorin response curve that lies between the minimum and maximum values of fluctuating calcium, and must lie above the curve. By explicitly constructing this region, we derive a quartic equation that gives the largest measured calcium for which aequorin can be assumed to give an overestimate, as a function of the maximum calcium fluctuation. In particular, if calcium fluctuations do not exceed 1 mM, aequorin measurements below 7.25 microM may be assumed to overestimate the true mean calcium.     

Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root

Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.     

Effect of anaesthetics on calcium-induced luminescence of aequorin

The effect of some general and local anaesthetics on the calcium-induced luminescence of aequorin was studied in vitro using a photomultiplier tube and recording technique. Purified aequorin (0.1 microliter) was injected into a 500 micron diameter porous cellulose acetate capillary tube containing 0.5 M KC1, 20 mM phosphate (pH 7.2) and calcium-EGTA buffers. The trapped aequorin was superfused with buffer solutions which sometimes contained anaesthetic (test) solutions. The results showed that some anaesthetics, e.g. urethane, etomidate and lignocaine, increased whereas others, e.g. methohexitone, thiopentone, decreased the light output (luminescence) of aequorin in constant ionized calcium and EGTA buffers. Similar results were produced by some non-anaesthetic drugs, e.g. glycerol, TEA, caffeine, etc. Concentration-response curves for calcium-dependent and -independent luminescence of aequorin showed that anaesthetics variously affected the aequorin response. Some anaesthetics, e.g. lignocaine, increased the maximum response while others, e.g. etomidate, increased the affinity (i.e. decreased EC50s) of aequorin to calcium ions without altering the slope, which remained at about 2. It was concluded that anaesthetics can either excite or depress aequorin luminescence, the effect being dependent on the type and the concentration used.     

A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana

Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo.     

Cross-talk between cAMP and calcium signalling in Aspergillus niger

Very little is known about cross-talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)-dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon-optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl-cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP-mediated, PKA-dependent phosphorylation. Further evidence for cross-talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar-apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA-dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.     

Changes in Intracellular Free Calcium Concentration during Illumination of Invertebrate Photoreceptors : Detection with Aequorin

Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Ca_i. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Ca_o reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Ca_o increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Ca_i may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Ca_i is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level.     

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.     

The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.     

Calcium ions are involved in the delay of plant cell cycle progression by abiotic stresses

Higher plants respond to environmental stresses by a sequence of reactions which include the reduction of growth by affecting cell division. It has been shown that calcium ions plays a role as a second messenger in mediating various defence responses under environmental stresses. In this study, the role of calcium ions on cell cycle progression under abiotic stresses has been examined in tobacco BY-2 suspension culture cells. Using synchronized BY-2 cells expressing the endogenous calcium sensor aequorin as experimental system, we could show that oxidative and hypoosmotic stress both induce an increase of intracellular calcium and cause a delay of the cell cycle. The inhibitory effect of these abiotic stress stimuli on cell cycle progression could be mimicked by increasing the intracellular calcium concentration via application of an external electrical field. Likewise, depletion of calcium ions in the culture medium suppressed the effect of the stimuli tested. These results demonstrate that calcium signalling is involved in the regulation of cell cycle progression in response to abiotic stress.     

Calcium transients in single adrenal chromaffin cells detected with aequorin

The effect of 55 mM K+ and nicotine on intracellular free calcium was monitored in bovine adrenal chromaffin cells microinjected with aequorin. In contrast to results with quin 2, which suggested that stimulation of chromaffin cells resulted in sustained rises in free calcium, aequorin measurements showed that 55 mM K+ and nicotine resulted in a transient (60-90 s) elevation of free calcium. The peak free calcium and duration of the transient elicited by nicotine were dose-dependent. The concentration of nicotine (10 microM) giving a maximal secretory response gave a peak rise in free calcium of up to 1 microM. 55 mM K+ which only releases 30% of the catecholamine released by 10 microM nicotine generated a calcium transient indistinguishable from that due to 10 microM nicotine. These results support the idea that nicotine agonists generate an alternative second messenger in addition to the rise in free calcium.     

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.     

Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation.

Cold shock elicits an immediate rise in cytosolic free calcium concentration ([Ca2+]cyt) in both chilling-resistant Arabidopsis and chilling-sensitive tobacco (Nicotiana plumbaginifolia). In Arabidopsis, lanthanum or EGTA caused a partial inhibition of both cold shock [Ca2+]cyt elevation and cold-dependent kin1 gene expression. This suggested that calcium influx plays a major role in the cold shock [Ca2+]cyt response and that an intracellular calcium source also might be involved. To investigate whether the vacuole (the major intracellular calcium store in plants) is involved, we targeted the calcium-dependent photoprotein aequorin to the cytosolic face of the vacuolar membrane. Cold shock calcium kinetics in this microdomain were consistent with a cold-induced vacuolar release of calcium. Treatment with neomycin or lithium, which interferes with phosphoinositide cycling, resulted in cold shock [Ca2+]cyt kinetics consistent with the involvement of inositol trisphosphate and inositide phosphate signaling in this response. We also investigated the effects of repeated and prolonged low temperature on cold shock [Ca2+]cyt. Differences were observed between the responses of Arabidopsis and N. plum-baginifolia to repeated cold stimulation. Acclimation of Arabidopsis by pretreatment with cold or hydrogen peroxide caused a modified calcium signature to subsequent cold shock. This suggests that acclimation involves modification of plant calcium signaling to provide a "cold memory."     

非生物逆境胁迫下植物钙信号转导的分子机制

Ca2+作为植物细胞中最重要的第二信使, 参与植物对许多逆境信号的转导。在非生物逆境条件下, 植物细胞质内的钙离子在时间、空间及浓度上会出现特异性变化, 即诱发产生钙信号。钙信号再通过其下游的钙结合蛋白进行感受和转导, 进而在细胞内引起一系列的生物化学反应以适应或抵制各种逆境胁迫。目前在植物细胞中发现Ca2+/CDPK、Ca2+/CaM和Ca2+/CBL 3类钙信号系统, 研究表明它们与非生物逆境胁迫信号转导密切相关。本文通过从植物在非生物逆境条件下钙信号的感受、转导到产生适应性和抗性等方面, 介绍钙信号转导分子机制的一些研究进展。     

Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli

We describe a novel and simple method for the measurement of bacterial cytosolic free calcium ([Ca2+]i) using recombinant aequorin reconstituted within live bacterial cells. Using this method we have measured the effects of external calcium, complement, phagocytosis and antibiotics on the [Ca2+]i of Escherichia coli. In principle this method should be applicable to any genetically transformable organism and should suffer fewer problems than fluorescent dyes for subcellular calcium measurement.