- freely available
Int. J. Mol. Sci. 2014, 15(3), 3842-3859; doi:10.3390/ijms15033842
Published: 4 March 2014
Abstract: The calcium ion (Ca2+) is a versatile intracellular messenger. It provides dynamic regulation of a vast array of gene transcriptions, protein kinases, transcription factors and other complex downstream signaling cascades. For the past six decades, intracellular Ca2+ concentration has been significantly studied and still many studies are under way. Our understanding of Ca2+ signaling and the corresponding physiological phenomenon is growing exponentially. Here we focus on the improvements made in the development of probes used for Ca2+ imaging and expanding the application of Ca2+ imaging in plant science research.
Knowledge of Ca2+ signaling and its corresponding physiological phenomenon from prokaryotes to eukaryotes and from tissues to whole organisms has grown significantly. For the past six decades, calcium signaling has been a focus of study with a level of investigation higher than that of any other signaling molecule. In plants, numerous endogenous stimuli and stress signals of both biotic and abiotic nature lead to transient variation in intracellular Ca2+ concentration, which in turn activate respective downstream signaling cascades [1–6]. Studying the role of intracellular Ca2+ requires the ability to monitor the dynamics of its concentration in plant cells with both spatial and temporal accuracy [7–9].
In plants, the concentration of cytosolic Ca2+ ([Ca2+]cyt) is maintained in the nano molar range (100–200 nM) while in many organelles it may reach micro molar to mili molar concentrations [10,11]. Vacuole and apoplast shows mili molar Ca2+ concentration . The use of new generation cameleon and aequorin (AEQ) has led to the discovery of Ca2+ dynamics at micor molar level in ER lumen and at nM level in mitochondria and peroxisomes [13–19]. In plant cells, the spatial and temporal dynamic changes of [Ca2+]cyt induced by various stimuli determine the final functional outcome [9,20]. Therefore, in plant cells, determining changes of [Ca2+]cyt is largely dependent on the development of methodologies that can be used to accurately measure [Ca2+]cyt [21,22].
Here, we describe the development of various Ca2+ probes from the past six decades and the improvements that have been developed in this field. We also discuss the limitations of each probe and important points to consider while planning ideal Ca2+ imaging experiments in plant science.
2. Measuring [Ca2+]cyt in Living Plant Cells
Confocal microscopy is an optical sectioning method used to reduce the image blur that is caused by inclusion of light from outside the plane-of-focus in a cross-section of a thick sample. In confocal microscopy, the path of out-of-focus light is physically blocked before detection . As a comparatively non-destructive imaging technique, confocal laser scanning microscopy (CLSM) has a number of distinct advantages over alternative imaging modalities; primarily CLSM facilitates the in situ characterization of the 3D architecture of tissue microstructure . Several papers addressed the benefits that CLSM affords during analysis of the spatial properties of intracellular [Ca2+] signals . However, it is generally difficult to measure [Ca2+] in a non-invasive method and without artifacts. It is also particularly tricky to measure [Ca2+] in a physiologically relevant context, which allows the comparison of results obtained in the laboratory with the physiological status of plants in the field. CLSM makes extensive use of Ca2+ probes. However, not only CLSM can offer the possibility to couple the use of Ca2+ probes with in vivo microscopy analyses. Wide-field fluorescent microscopy has been used for both dyes and genetically encoded probes. [Ca2+]cyt was determined using the dye Fura-2 by either fluorescence ratio with a Cairn micro photometer  or fluorescence ratio imaging with a GenIV-intensified Pentamax-512 charge-coupled device camera . By using high-resolution deconvolution microscopy, Allen and co-workers  assessed the cytoplasmic localization and auto fluorescence at both emission wavelengths of cameleon, YC2.1. Recently, Costa and co-workers used Selective Plane Illumination Microscopy (SPIM) which is an imaging technique particularly suited for long term in vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. SPIM was successfully used in calcium imaging based on Förster Resonance Energy Transfer (FRET) in Arabidopsis seedlings expressing the cytosolic (NES-YC3.6) or nuclear (NLS-YC3.6) localized Cameleon YC3.6 .
3. Origin and Evolution of Synthetic Ca2+ Indicators
In the early 1960s to 1970s, many synthetic indicators such as murexide, azo dyes, and chlortetracycline were used as Ca2+ indicators . Among the limitations of these synthetic indicators are a low sensitivity (azo dyes), the impossibility to be used to measure Ca2+ in living cells (murexide) and a low accuracy (chlorotetracycline) . The first fluorescent Ca2+ probe ideal for intracellular Ca2+ measurement was synthesized by Roger Tsien in the late 1980s . Later on, a range of synthetic dyes was developed with desirable sensitivity, selectivity and responsiveness to measure Ca2+ in living plant cells [8,21]. Moreover, the evolution of AEQ [31,32], green fluorescent protein (GFP)  and FRET [34,35] based Ca2+ fluorescent imaging became more popular because of their user friendly Ca2+ measurement methods; however, Ca2+ fluorescent dyes have the advantages of being applicable to plants not suitable for transformation.
4. Measurement of Ca2+ Using Non-Ratiometric Dyes or Single Wavelength Probes
Ca2+ probes (also referred as sensors or reporters) are small molecules that show desirable features of sensitivity, selectivity and responsiveness to Ca2+. These probes form selective complexes with Ca2+ ions, which enable measurement of the differences in free and bound [Ca2+] using absorbance and emission light [36–38]. Most of the Ca2+ imaging probes interact with Ca2+ through carboxylic acid groups. This interaction causes variation in indicator properties such as fluorescent intensity and its excitation. [Ca2+] is not measured directly; rather the indicator monitors the amount of free and complexed probe. The concentration of free Ca2+ is then measured based on effective dissociation constant (Kd) measured in vitro of the probe for Ca2+ in the specific environment.
[Ca2+]cyt is measured based on relative increase in fluorescence intensity of single wavelength. Non-ratiometric dyes such as fluo, rhod and Calcium Green-1 are used for measuring [Ca2+]cyt in plants, among them Calcium Green-11 is well-known and most commonly used in plant systems. The single excitation spectrum allows for simple instrumentation. A simple formula is used to calculate the absolute Ca2+ concentration in live plant cells.
where Kd is the dissociation constant for Ca2+ for the fluorescent dye which is measured in vitro; F stands for fluorescence measured; Fmin is the fluorescence of the probe in the absence of Ca2+ and Fmax is the fluorescence of the probe at saturation point . Calibration of absolute Ca2+ in in vitro samples are much easier using this method compared to ratiometric dyes but in vivo measurement in plant cells is a challenging task compared to animal cells. In an animal cell, it is easier to use mild detergent to permeabilize the entry of the Ca2+ dye through the plasma membrane, but in plants, the cell wall forms a major barrier. In plant and animal cells, limitations of this method are: corrections cannot be made for photobleaching, unequal dye loading, movements of dye within the cell and changes in optical density of the cell which affects the fluorescent intensity and accurate measurement of Ca2+. Moreover, these dyes are sensitive to pH, ionic strength and the surrounding protein environment, which alters their properties [7,8,21]. In order to overcome these limitations, it is recommended to simultaneously collect information on Ca2+-dependent fluorescence and probe concentration . Other limitations such as optical artifacts could be overcome by using ratiometric dyes. These dyes offer robust approaches for in vivo quantitative measurement of Ca2+ based on fluorescence changes.
5. Measurement of Ca2+ Using Ratiometric Dyes
The excitation spectrum of ratiometric dyes Fura-2 and Indo-1, varies according to the free Ca2+ concentration. The Ca2+ concentration is measured as the ratio between two fluorescence intensity values that are taken at two wavelengths; i.e., increasing wavelength λ1 and decreasing wavelength λ2. The absolute [Ca2+] can be calculated using the following formula:
where Kd is the dissociation constant, which is measured in vitro; R is the fluorescence ratio at both wavelengths Fλ2/Fλ1; Rmin is the minimum ratio value (it can be at minimum or maximum calcium concentration, depending on the wavelength used in the denominator); Rmax is the maximum ratio value; is a scaling factor, also known as β (Fλ1 is the fluorescence used in the denominator at its maximum and minimum value).
Ratiometric dyes can be corrected for unequal dye loading, photobleaching and focal plane shift, between two cells that have the same [Ca2+]cyt. Therefore using ratios avoids many of the problems related to absolute fluorescence values. On the other hand, acquisition and data manipulation is more complex due to the use of fluorescence ratios. Not all microscopes are suitable for these measurements (changing excitation/emission wavelength at suitable rates is required) and many ratiometric indicators require the use of UV excitation, which is an expensive option for confocal laser scanning microscopy.
It is important to note that dyes are available at different ranges of Kd values and spectral properties, that make them suitable for commonly used lasers .
6. Techniques of Loading Ca2+-Sensing Dyes into Plant Cells
Most of the Ca2+ interacting dyes used for Ca2+ measurement have highly charged groups, hence they are relatively membrane impermeable; therefore, loading probes in living plant cells is a challenging task . Many loading techniques have been successfully adopted to breach the plasma membrane such as microinjection, electroporation, patch clump pipette and biolistic delivery [41–44]. Acid and ester methods are also adopted to introduce Ca2+ dyes; the acid method is based on pH changes. Acid loading for a 1–2 h incubation causes a drop in pH to 4.5 and protonates the dye to breach the plasma membrane in a relatively uncharged form [45,46]. Samples are incubated for 1–2 h at low pH, which might cause adverse effects on plant cells and tissues. In case of ester loading, acetoxymethyl esters are generally used for intracellular cellular cleavage by cellular esterases to deliver the Ca2+ dye into cytosol . Ester loading causes significant hydrolysis in the plant cell wall but loading and incubation at low temperature may limit this problem . Another major problem of Ca2+ interacting dyes is their compartmentalization in cellular organelles, mainly in the vacuole [45,48]. Compartmentalization means that the indicator is trapped within cellular organelles and not homogenously distributed throughout the plant cell .
It is hard to indicate which is the most efficient or the most effective dye. This mainly depends on the overlapping of emissions with autofluorescence from the tissue or the ability of the dye to penetrate the plant tissues. For example, in a recent paper, Fluo-3, which was successfully used for the calcium localization in Lima bean , was found to strongly interfere with the cuticular autoflourescence of Ginkgo biloba leaves. In this case, Calcium Orange was selected because of a different fluorescence emission .
Table 1 lists some of the most used Ca2+ dyes.
7. Protein Based Ca2+ Indicators
7.1. Aequorin–Based Ca2+ Indicators
Aequorin (AEQ) photoprotein has been extensively used in the Ca2+ signaling field for almost 40 years. AEQ is a Ca2+-binding photoprotein composed of an apoprotein (apoaequorin), which has an approximate molecular weight of 22 kDa and a prosthetic group, a luciferin molecule, coelenterazine (Mr 432). In the presence of molecular oxygen, the functional holoprotein, aequorin, reconstitutes spontaneously. The protein contains three EF-hand Ca2+-binding sites. When these sites are occupied by Ca2+, aequorin undergoes a conformational change and behaves as an oxygenase that converts coelenterazine into excited coelenteramide, which is released together with carbon dioxide. When the excited coelenteramide relax to its ground state, blue light (λ = 469 nm) is emitted . This emitted light can be easily detected with a luminometer and correlates with the particular [Ca2+]. Identification of Ca2+ sensitive AEQ from Aequorea victoria offers enormous advantage to carry out bioluminescence research. This protein was carefully extracted and purified from jelly fish to prevent the contact with Ca2+ as this would cause chemiluminescence, there by rendering the protein unsuitable for Ca2+ measurements . These proteins have been extensively engineered to obtain several luminescent probes with different biological parameters [62–64]. With the advanced genetic engineering and cloning strategies, it is possible to specifically localize them within the cell by including specific targeting amino acid sequences .
In the last two decades the study of Ca2+ dynamics in living cells has been enhanced by a significant improvement of genetically encoded protein based indicators . AEQ allows their endogenous production in cell system as diverse as bacteria, yeast, fungi, plants and mammalian cells.
The new generation of bioluminescent probes coupled with Ca2+ sensitive AEQ allows real time measurement of Ca2+ changes [65,66]. Recently, AEQ based luminescent recording system has been developed to monitor spatiotemporal Ca2+ dynamics to various stimuli in Arabidopsis plants .
A major advantage of AEQ is that it can be selectively targeted to subcellular compartments by insertion of signal sequences unlike chemical compound dyes (excluding rhod-2, which is largely retained in the mitochondrial matrix). Use of recombinant AEQ facilitated the understanding of Ca2+ signaling interplay between different cellular compartments [67–72]. AEQ became less popular because of its small inherent signal; although the amount of signal emitted by the cell population is quite adequate to measure Ca2+ concentration, the signal from single cell is very low. Adequate quantity of signal is needed to overcome background noise at the expense of space and time resolutions. One of the limitations of AEQ use is that it usually overestimates the real response of cells, especially when cell suspensions are used instead of whole tissues. For instance, when comparing Fluo-3AM responses in Lima bean leaf tissues and AEQ signals in soybean cell suspensions, the upper level of estimated level of Ca2+ in soybean cell culture responsiveness to H2O2 corresponds to the lower level in both mechanically damaged and herbivore wounded Lima bean leaves. This situation reflects the higher sensitivity of cell suspensions cultures compared to plant tissues, and should be considered for further comparisons when AEQ is used to evaluate activities of molecules involved in signaling processes . However, not only suspension cell cultures expressing AEQ have been used, but also seedlings or leaf discs. For example, Matrí and co-workers  used GAL4 transactivation of AEQ to analyze [Ca2+]cyt signaling in specific cell types, including those of the leaves. Therefore, AEQ can offer tissue specificity if placed under control of tissue-specific promoters.
In AEQ assays the emitted light is calibrated into Ca2+ concentrations by a method based on the calibration curve of Allen and coworkers :
where L0 is the luminescence intensity per second and Lmax is the total amount of luminescence present in the entire sample over the course of the experiment. [Ca2+] is the calculated Ca2+ concentration, KR is the dissociation constant for the first Ca2+ ion to bind, and KTR is the binding constant of the second Ca2+ ion to bind to AEQ . Figure 1 shows the principle of the AEQ reaction.
7.2. GFP–Based Ca2+ Indicators
GFP based Ca2+ sensors are immediate alternatives to synthetic dyes and AEQ described above. In 1997 the first GFP based recombinant Ca2+ probe was developed [33,76]. Even though there are many indicators, a limited number of those have been used in plants . Currently there are three main types of this Ca2+ sensor: cameleons, camgaroos and pericams [61,78,79]. All these sensors were based on calmodulin (CaM) as a regulator, which changes its confirmation and alters fluorescence properties upon binding to Ca2+. The cameleon probe has been extensively used in plant science research as compared to camagaroos and pericams [7,8,21,27]. These probes are chimeric proteins designed on the property called change of FRET, first synthesized by Tsien and co-workers [79,80]. FRET occurs between two different colored GFP mutants, with spectral overlap of the donor emission spectrum and the acceptor absorption spectrum. In the probe, the two GFP mutants are linked together by CaM and CaM binding peptide . Binding of Ca2+ to the Ca2+- responsive elements alters the efficiency of FRET. Like AEQ, several generations of FRET-based Ca2+ were synthesized with different biological parameters, sensitivity and efficiency; among them yellow cameleon (YC2.1) is the most popular for their use under different physiological conditions [80,81]. The YC2.1 version of cameleon has been widely used in plants for measurements of Ca2+ fluxes in guard cells, Nod factor responses, rhizobium and fungal colonization in the roots [27,82–85]. Later on various modified version of cameleon sensors were used to dissect subcellular Ca2+ dynamics; for example, D3cpv cameleon sensor for peroxisome Ca2+ flux and YC4.6 for pollen tube endoplasmic reticulum Ca2+ dynamics [17,19]. Two recent works report the use of D3cpv for mitochondrial Ca2+ analysis and D4ER for ER Ca2+ analysis [14,15]. Considering their importance in measuring Ca2+ dynamics at subcellular levels, efforts were made to develop a new generation of YC2.1 with significantly increased FRET signal to provide efficient Ca2+ measurements while reducing signal to noise ratio, this modified version was named YC3.6 [61,79,86]. This new generation with high-resolution signal has been widely used in plants for spatiotemporal imaging of cytoplasmic Ca2+ fluxes [87,88]. Recently YC3.6 has been successfully used to measure the cytosolic Ca2+ upon mechanical damage and herbivory in Arabidopsis leaves . A wide range of YC sensors were successfully employed in plant science to analyze the spatiotemporal Ca2+ flux in different cell types such as guard cells, root, root hairs, pollen tube and different subcellular compartments [13,17,90–92]. Successes of the cameleon-based sensors are limited by CaM binding peptide as part of the sensing mechanism. Endogenous CaM could interfere with the sensor and could possibly change FRET signal.
Leaves of plants expressing FRET-based Ca2+ sensor YC3.6 can be ratio-imaged by CLSM. The YC3.6 Ca2+ sensor is usually excited at 458-nm wavelength by using an argon laser. The cyan fluorescent protein (CFP) and FRET-dependent Venus emission are assayed using a krypton/argon laser at 458 nm with a 473–505 and 526–536 nm emission filters. In situ calibration is performed through raising Ca2+ to saturating levels for YC3.6. Cells are usually permeabilized to allow a massive free diffusion of calcium inside the cell to get the Rmax. EGTA and EDTA can trap the free calcium released from the cells. Therefore, the maximum FRET/CFP ratio is obtained by treatment with 1 molar CaCl2 in response to mechanical perturbation. The minimum FRET/CFP ratio is recorded by treatment with 1 molar Tris 100 mM EDTA and 50 mM EGTA solution. [Ca2+]cyt variations are then calculated according to the equation:
where R represents the FRET/CFP ratio measured during the experiment, n the Hill coefficient for YC3.6 (usually = 1), while Kd values are assessed for a given concentration of Ca2+ . Figure 2 illustrates the determination of [Ca2+]cyt variations using a cameleon probe.
Table 2 lists some of the most used GFP-based Ca2+ indicators in plant sciences.
8. Conclusions and Future Perspectives
Imaging Ca2+ in living cells has seen a tremendous development in the last two decades with the evolution of genetically encoded Ca2+ indicators. Despite these improvements, we are still far from having an ideal Ca2+ probe. Here we give emphasis on future directions on the improvements of synthetic and fluorescent Ca2+ probes.
Currently, there are only two ratiomatric dyes fura-2 and indo-1 available since thirty years after their synthesis. Both these dyes requires UV spectrum for their excitation. As a result, there is the need for the use of expensive UV lasers for confocal microscopes, with the results of toxicity of UV illumination, lower penetration and background fluorescence. Therefore, it is necessary to develop new indicators that absorb light in the visible range.
The advent of a new generation of protein-based Ca2+ indicators has reduced the demand to develop new optimized dyes. Although these protein-based probes offer many advantages over synthetic dyes they are still far from being ideal probes. Most of the GFP-based probes have CaM as Ca2+ sensing component. CaMs are also known to be important for many physiological processes in plant system. Overexpression of GFP-based probes might results in the substantial alteration of endogenous physiological processes that depend on CaM activity [81,102]. Therefore, efforts should focus on designing new protein-based constructs as selective and sensitive as CaM but characterized by a lower or negligible interference with the endogenous components of the plant cell . Further improvements on GFP-based probes should face the problem of bleaching and photoisomerization of GFP upon illumination . Finding novel fluorescent protein from other organisms (other than Aeuuorea) would provide solutions to these problems. All these efforts will allow us to understand the complex subcellular interplay of Ca2+ signals underlying many plant physiological and developmental processes.
The research leading to the results reported here has received funding from the Autonomous Province of Trento, Italy, with reference to the Call 1-post-doc 2012–Incoming, approved with provincial government resolution No. 1023 of 5/7/10.
Conflicts of Interest
The authors declare no conflict of interest.
- Whalley, H.J.; Knight, M.R. Calcium signatures are decoded by plants to give specific gene responses. New Phytol. 2013, 197, 690–693, doi:10.1111/nph.12087. 23190495
- Arimura, G.; Maffei, M.E. Calcium and secondary CPK signaling in plants in response to herbivore attack. Biochem. Biophys. Res. Commun. 2010, 400, 455–460, doi:10.1016/j.bbrc.2010.08.134. 20816754
- McAinsh, M.R.; Pittman, J.K. Shaping the calcium signature. New Phytol. 2009, 181, 275–294, doi:10.1111/j.1469-8137.2008.02682.x. 19121028
- White, P.J.; Broadley, M.R. Calcium in plants. Ann. Bot. 2003, 92, 487–511, doi:10.1093/aob/mcg164. 12933363
- Monshausen, G.B. Visualizing Ca(2+) signatures in plants. Curr. Opin. Plant Biol. 2012, 15, 677–682, doi:10.1016/j.pbi.2012.09.014. 23044039
- Quiles-Pando, C.; Rexach, J.; Navarro-Gochicoa, M.T.; Camacho-Cristobal, J.J.; Herrera-Rodriguez, M.B.; Gonzalez-Fontes, A. Boron deficiency increases the levels of cytosolic Ca(2+) and expression of Ca(2+)-related genes in Arabidopsis thaliana roots. Plant Physiol. Biochem. 2013, 65, 55–60, doi:10.1016/j.plaphy.2013.01.004. 23416496
- Swanson, S.J.; Gilroy, S. Imaging changes in cytoplasmic calcium using the Yellow Cameleon 36 biosensor and confocal microscopy. Methods Mol. Biol. 2013, 1009, 291–302. 23681544
- Choi, W.G.; Swanson, S.J.; Gilroy, S. High-resolution imaging of Ca2+ redox status ROS, and pH using GFP biosensors. Plant J. 2012, 70, 118–128, doi:10.1111/j.1365-313X.2012.04917.x. 22449047
- Batistic, O.; Kudla, J. Analysis of calcium signaling pathways in plants. Biochim. Biophys. Acta 2012, 1820, 1283–1293, doi:10.1016/j.bbagen.2011.10.012. 22061997
- Dodd, A.N.; Kudla, J.; Sanders, D. The language of calcium signaling. Ann. Rev. Plant Biol. 2010, 61, 593–620, doi:10.1146/annurev-arplant-070109-104628.
- McAinsh, M.R.; Ng, C.K. Measurement of cytosolic-free Ca2+ in plant tissue. Methods Mol. Biol. 2013, 937, 327–341. 23007596
- Stael, S.; Wurzinger, B.; Mair, A.; Mehlmer, N.; Vothknecht, U.C.; Teige, M. Plant organellar calcium signalling: An emerging field. J. Exp. Bot. 2012, 63, 1525–1542, doi:10.1093/jxb/err394. 22200666
- Behera, S.; Krebs, M.; Loro, G.; Schumacher, K.; Costa, A.; Kudla, J. Ca2+ imaging in plants using genetically encoded Yellow Cameleon Ca2+ indicators. Cold Spring Harb. Protoc. 2013, 2013, 700–703. 23906906
- Loro, G.; Ruberti, C.; Zottini, M.; Costa, A. The D3cpv Cameleon reports Ca2+ dynamics in plant mitochondria with similar kinetics of the YC36 Cameleon but with a lower sensitivity. J. Microsc. 2013, 249, 8–12, doi:10.1111/j.1365-2818.2012.03683.x. 23227874
- Bonza, M.C.; Loro, G.; Behera, S.; Wong, A.; Kudla, J.; Costa, A. Analyses of Ca2+ accumulation and dynamics in the endoplasmic reticulum of Arabidopsis root cells using a genetically encoded Cameleon sensor. Plant Physiol. 2013, 163, 1230–1241, doi:10.1104/pp.113.226050. 24082028
- Loro, G.; Drago, I.; Pozzan, T.; Schiavo, F.L.; Zottini, M.; Costa, A. Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca2+ handling relationship in plant cells. Plant J. 2012, 71, 1–13, doi:10.1111/j.1365-313X.2012.04968.x. 22372377
- Costa, A.; Drago, I.; Behera, S.; Zottini, M.; Pizzo, P.; Schroeder, J.I.; Pozzan, T.; Lo Schiavo, F. H2O2 in plant peroxisomes: An in vivo analysis uncovers a Ca2+-dependent scavenging system. Plant J. 2010, 62, 760–772, doi:10.1111/j.1365-313X.2010.04190.x. 20230493
- Logan, D.C.; Knight, M.R. Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants. Plant Physiol. 2003, 133, 21–24, doi:10.1104/pp.103.026047. 12970470
- Iwano, M.; Entani, T.; Shiba, H.; Kakita, M.; Nagai, T.; Mizuno, H.; Miyawaki, A.; Shoji, T.; Kubo, K.; Isogai, A.; et al. Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth. Plant Physiol. 2009, 150, 1322–1334, doi:10.1104/pp.109.139329. 19474213
- Mithofer, A.; Mazars, C.; Maffei, M.E. Probing Spatio-temporal Intracellular Calcium Variations in Plants. Methods Mol. Biol. 2009, 479, 79–92. 19083174
- Swanson, S.J.; Choi, W.G.; Chanoca, A.; Gilroy, S. In vivo imaging of Ca2+ pH and reactive oxygen species using fluorescent probes in plants. Ann. Rev. Plant Biol. 2011, 62, 273–297, doi:10.1146/annurev-arplant-042110-103832.
- Joy, M.J.; Pyniarlang, N.; Ralf, O.; Irena, S. Calcium signaling and cytosolic calcium measurements in plants. J. Endocytobiosis Cell Res. 2011, 21, 64–76.
- Reddy, G.V.; Gordon, S.P.; Meyerowitz, E.M. Unravelling developmental dynamics: Transient intervention and live imaging in plants. Nat. Rev. Mol. Cell Biol. 2007, 8, 491–501, doi:10.1038/nrm2188. 17522592
- Foldes-Papp, Z.; Demel, U.; Tilz, G.P. Laser scanning confocal fluorescence microscopy: An overview. Int. Immunopharmacol. 2003, 3, 1715–1729, doi:10.1016/S1567-5769(03)00140-1. 14636823
- Kohler, B.; Blatt, M.R. Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid. Plant J. 2002, 32, 185–194, doi:10.1046/j.1365-313X.2002.01414.x. 12383084
- Garcia-Mata, C.; Gay, R.; Sokolovski, S.; Hills, A.; Lamattina, L.; Blatt, M.R. Nitric oxide regulates K+ and Cl− channels in guard cells through a subset of abscisic acid-evoked signaling pathways. Proc. Natl. Acad. Sci. USA 2003, 100, 11116–11121, doi:10.1073/pnas.1434381100. 12949257
- Allen, G.J.; Kwak, J.M.; Chu, S.P.; Llopis, J.; Tsien, R.Y.; Harper, J.F.; Schroeder, J.I. Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J. 1999, 19, 735–747, doi:10.1046/j.1365-313x.1999.00574.x. 10571859
- Costa, A.; Candeo, A.; Fieramonti, L.; Valentini, G.; Bassi, A. Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy. PLoS One 2013, 8, e75646, doi:10.1371/journal.pone.0075646. 24146766
- Tsien, R.Y. New calcium indicators and buffers with high selectivity against magnesium and protons: design synthesis and properties of prototype structures. Biochemistry 1980, 19, 2396–2404, doi:10.1021/bi00552a018. 6770893
- Rudolf, R.; Mongillo, M.; Rizzuto, R.; Pozzan, T. Looking forward to seeing calcium. Nat. Rev. Mol. Cell Biol. 2003, 4, 579–586, doi:10.1038/nrm1153. 12838340
- Shimomura, O.; Johnson, F.H.; Saiga, Y. Extraction purification and properties of aequorin a bioluminescent protein from the luminous hydromedusan Aequorea. J. Cell Comp. Physiol. 1962, 59, 223–239, doi:10.1002/jcp.1030590302. 13911999
- Knight, M.R.; Read, N.D.; Campbell, A.K.; Trewavas, A.J. Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins. J. Cell Biol. 1993, 121, 83–90, doi:10.1083/jcb.121.1.83. 8458875
- Miyawaki, A.; Llopis, J.; Heim, R.; McCaffery, J.M.; Adams, J.A.; Ikura, M.; Tsien, R.Y. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 1997, 388, 882–887, doi:10.1038/42264. 9278050
- Jares-Erijman, E.A.; Jovin, T.M. FRET imaging. Nat. Biotechnol. 2003, 21, 1387–1395, doi:10.1038/nbt896. 14595367
- Sekar, R.B.; Periasamy, A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 2003, 160, 629–633, doi:10.1083/jcb.200210140. 12615908
- Arimura, G.; Kopke, S.; Kunert, M.; Volpe, V.; David, A.; Brand, P.; Dabrowska, P.; Maffei, M.E.; Boland, W. Effects of feeding Spodoptera littoralis on lima bean leaves: IV Diurnal and nocturnal damage differentially initiate plant volatile emission. Plant Physiol. 2008, 146, 965–973, doi:10.1104/pp.107.111088. 18165324
- Kanchiswamy, C.N.; Mohanta, T.K.; Capuzzo, A.; Occhipinti, A.; Verrillo, F.; Maffei, M.E.; Malnoy, M. Differential expression of CPKs and cytosolic Ca2+ variation in resistant and susceptible apple cultivars (Malus × domestica) in response to the pathogen Erwinia amylovora and mechanical wounding. BMC Genomics 2013, 14, 760, doi:10.1186/1471-2164-14-760. 24192013
- Kanchiswamy, C.N.; Takahashi, H.; Quadro, S.; Maffei, M.E.; Bossi, S.; Bertea, C.; Zebelo, S.A.; Muroi, A.; Ishihama, N.; Yoshioka, H.; et al. Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling. BMC Plant Biol. 2010, 10, 97, doi:10.1186/1471-2229-10-97. 20504319
- Grynkiewicz, G.; Poenie, M.; Tsien, R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 1985, 260, 3440–3450. 3838314
- Pierson, E.S.; Miller, D.D.; Callaham, D.A.; Shipley, A.M.; Rivers, B.A.; Cresti, M.; Hepler, P.K. Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell 1994, 6, 1815–1828. 7866026
- Bothwell, J.H.; Brownlee, C.; Hetherington, A.M.; Ng, C.K.; Wheeler, G.L.; McAinsh, M.R. Biolistic delivery of Ca2+ dyes into plant and algal cells. Plant J. 2006, 46, 327–335, doi:10.1111/j.1365-313X.2006.02687.x. 16623894
- Romano, L.A.; Jacob, T.; Gilroy, S.; Assmann, S.M. Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L. Planta 2000, 211, 209–217, doi:10.1007/s004250000286. 10945215
- Zebelo, S.A.; Matsui, K.; Ozawa, R.; Maffei, M.E. Plasma membrane potential depolarization and cytosolic calcium flux are early events involved in tomato (Solanum lycopersicon) plant-to-plant communication. Plant Sci. Biol. 2012, 196, 93–100.
- Bricchi, I.; Occhipinti, A.; Bertea, C.M.; Zebelo, S.A.; Brillada, C.; Verrillo, F.; de Castro, C.; Molinaro, A.; Faulkner, C.; Maule, A.J.; et al. Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function. Plant J. 2012, doi:10.1111/j.1365-313X. 2012.05103.x.
- Bush, D.S. Regulation of cytosolic calcium in plants. Plant Physiol. 1993, 103, 7–13. 12231912
- Dauphin, A.; Gerard, J.; Lapeyrie, F.; Legue, V. Fungal hypaphorine reduces growth and induces cytosolic calcium increase in root hairs of Eucalyptus globulus. Protoplasma 2007, 231, 83–88, doi:10.1007/s00709-006-0240-9. 17370110
- Zhang, W.-H.; Rengel, Z.; Kuo, J. Determination of intracellular Ca2+ in cells of intact wheat roots: Loading of acetoxymethyl ester of Fluo-3 under low temperature. Plant J. 1998, 15, 147–151, doi:10.1046/j.1365-313X.1998.00188.x.
- Bush, D.S.; Biswas, A.K.; Jones, R.L. Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels. Planta 1989, 178, 411–420, doi:10.1007/BF00391870. 24212909
- Maffei, M.; Bossi, S.; Spiteller, D.; Mithofer, A.; Boland, W. Effects of feeding Spodoptera littoralis on lima bean leaves I Membrane potentials intracellular calcium variations oral secretions and regurgitate components. Plant Physiol. 2004, 134, 1752–1762, doi:10.1104/pp.103.034165. 15051862
- Mohanta, T.K.; Occhipinti, A.; Zebelo, S.A.; Foti, M.; Fliegmann, J.; Bossi, S.; Maffei, M.E.; Bertea, C.M. Ginkgo biloba responds to herbivory by activating early signaling and direct defenses. PLoS One 2012, 7, e32822, doi:10.1371/journal.pone.0032822. 22448229
- Qu, H.; Jiang, X.; Shi, Z.; Liu, L.; Zhang, S. Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia. J. Plant Res. 2012, 125, 185–195, doi:10.1007/s10265-011-0440-z. 21789557
- Zhao, X.; Wang, Y.J.; Wang, Y.L.; Wang, X.L.; Zhang, X. Extracellular Ca2+ alleviates NaCl-induced stomatal opening through a pathway involving H2O2-blocked Na+ influx in Vicia guard cells. J. Plant Physiol. 2011, 168, 903–910, doi:10.1016/j.jplph.2010.11.024. 21367483
- Liu, G.; Dang, L.; Wang, D.M. The influence of elicitor on the distribution pattern of microtubule and the cytosolic calcium in mesophyll protoplast of wheat. Fen Zi Xi Bao Sheng Wu Xue Bao 2007, 40, 205–213. 17966457
- Nair, R.; Raina, S.; Keshavarz, T.; Kerrigan, M.J. Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi. Fungal Biol. 2011, 115, 326–334, doi:10.1016/j.funbio.2010.12.012. 21530914
- Schulz, A.; Woolley, R.; Tabarin, T.; McDonagh, C. Dextran-coated silica nanoparticles for calcium-sensing. Analyst 2011, 136, 1722–1727, doi:10.1039/c0an01009j. 21369598
- Wu, X.; Chen, T.; Zheng, M.; Chen, Y.; Teng, N.; Samaj, J.; Baluska, F.; Lin, J. Integrative proteomic and cytological analysis of the effects of extracellular Ca2+ influx on Pinus bungeana pollen tube development. J. Proteome Res. 2008, 7, 4299–4312, doi:10.1021/pr800241u. 18715029
- Lu, Y.; Paige, M.F. An ensemble and single-molecule fluorescence spectroscopy investigation of Calcium Green 1 a calcium-ion sensor. J. Fluoresc. 2007, 17, 739–748, doi:10.1007/s10895-007-0185-1. 17638063
- Bagh, S.; Paige, M.F. Ensemble and single-molecule fluorescence spectroscopy of a calcium-ion indicator dye. J. Phys. Chem. A 2006, 110, 7057–7066, doi:10.1021/jp060719e. 16737253
- Braun, M.; Richter, P. Relocalization of the calcium gradient and a dihydropyridine receptor is involved in upward bending by bulging of Chara protonemata but not in downward bending by bowing of Chara rhizoids. Planta 1999, 209, 414–423, doi:10.1007/s004250050744. 10550622
- Bunney, T.D.; Shaw, P.J.; Watkins, P.A.; Taylor, J.P.; Beven, A.F.; Wells, B.; Calder, G.M.; Drobak, B.K. ATP-dependent regulation of nuclear Ca2+ levels in plant cells. FEBS Lett. 2000, 476, 145–149, doi:10.1016/S0014-5793(00)01709-9. 10913602
- Baird, G.S.; Zacharias, D.A.; Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 1999, 96, 11241–11246, doi:10.1073/pnas.96.20.11241. 10500161
- Ottolini, D.; Cali, T.; Brini, M. Measurements of Ca2+ concentration with recombinant targeted luminescent probes. Methods Mol. Biol. 2013, 937, 273–291. 23007593
- Zhu, X.; Feng, Y.; Liang, G.; Liu, N.; Zhu, J.K. Aequorin-based luminescence imaging reveals stimulus- and tissue-specific Ca2+ dynamics in Arabidopsis plants. Mol. Plant 2013, 6, 444–455, doi:10.1093/mp/sst013. 23371933
- Knight, H.; Knight, M.R. Recombinant aequorin methods for intracellular calcium measurement in plants. Methods Cell Biol. 1995, 49, 201–216. 8531755
- Brini, M.; Cali, T.; Ottolini, D.; Carafoli, E. Intracellular calcium homeostasis and signaling. Met. Ions Life Sci. 2013, 12, 119–168. 23595672
- Cali, T.; Ottolini, D.; Brini, M. Calcium and endoplasmic reticulum-mitochondria tethering in neurodegeneration. DNA Cell Biol. 2013, 32, 140–146, doi:10.1089/dna.2013.2011. 23477673
- Bonora, M.; Giorgi, C.; Bononi, A.; Marchi, S.; Patergnani, S.; Rimessi, A.; Rizzuto, R.; Pinton, P. Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes. Nat. Protoc. 2013, 8, 2105–2118, doi:10.1038/nprot.2013.127. 24113784
- Pozzan, T.; Rizzuto, R. Imaging calcium dynamics using targeted recombinant aequorins. CSH Protoc. 2008, 2008, doi:10.1101/pdb.top26.
- Rizzuto, R.; Pinton, P.; Brini, M.; Chiesa, A.; Filippin, L.; Pozzan, T. Mitochondria as biosensors of calcium microdomains. Cell Calcium 1999, 26, 193–199, doi:10.1054/ceca.1999.0076. 10643557
- Brini, M.; Pinton, P.; Pozzan, T.; Rizzuto, R. Targeted recombinant aequorins: Tools for monitoring [Ca2+] in the various compartments of a living cell. Microsc. Res. Tech. 1999, 46, 380–389, doi:10.1002/(SICI)1097-0029(19990915)46:6<380::AID-JEMT6>3.0.CO;2-Y. 10504215
- Pinton, P.; Brini, M.; Bastianutto, C.; Tuft, R.A.; Pozzan, T.; Rizzuto, R. New light on mitochondrial calcium. BioFactors 1998, 8, 243–253, doi:10.1002/biof.5520080312. 9914826
- Pinton, P.; Pozzan, T.; Rizzuto, R. The Golgi apparatus is an inositol 145-trisphosphatesensitive Ca2+ store with functional properties distinct from those of the endoplasmic reticulum. EMBO J. 1998, 17, 5298–5308, doi:10.1093/emboj/17.18.5298. 9736609
- Maffei, M.E.; Mithofer, A.; Arimura, G.; Uchtenhagen, H.; Bossi, S.; Bertea, C.M.; Cucuzza, L.S.; Novero, M.; Volpe, V.; Quadro, S.; et al. Effects of feeding Spodoptera littoralis on lima bean leaves III Membrane depolarization and involvement of hydrogen peroxide. Plant Physiol. 2006, 140, 1022–1035, doi:10.1104/pp.105.071993. 16443697
- Marti, M.C.; Stancombe, M.A.; Webb, A.A. Cell- and stimulus type-specific intracellular free Ca2+ signals in Arabidopsis. Plant Physiol. 2013, 163, 625–634, doi:10.1104/pp.113.222901. 24027243
- Allen, D.G.; Blinks, J.R.; Prendergast, F.G. Aequorin luminescence: Relation of light emission to calcium concentration—A calcium-independent component. Science 1977, 195, 996–998, doi:10.1126/science.841325. 841325
- Romoser, V.A.; Hinkle, P.M.; Persechini, A. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence A new class of fluorescent indicators. J. Biol. Chem. 1997, 272, 13270–13274, doi:10.1074/jbc.272.20.13270. 9148946
- Whitaker, M. Genetically encoded probes for measurement of intracellular calcium. Methods Cell Biol. 2010, 99, 153–182. 21035686
- Tozawa, Y.; Nozawa, A.; Kanno, T.; Narisawa, T.; Masuda, S.; Kasai, K.; Nanamiya, H. Calcium-activated (p)ppGpp synthetase in chloroplasts of land plants. J. Biol. Chem. 2007, 282, 35536–35545, doi:10.1074/jbc.M703820200. 17938177
- Nagai, T.; Sawano, A.; Park, E.S.; Miyawaki, A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 2001, 98, 3197–3202, doi:10.1073/pnas.051636098. 11248055
- Miyawaki, A.; Griesbeck, O.; Heim, R.; Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 1999, 96, 2135–2140, doi:10.1073/pnas.96.5.2135. 10051607
- Palmer, A.E.; Tsien, R.Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 2006, 1, 1057–1065, doi:10.1038/nprot.2006.172. 17406387
- Allen, G.J.; Chu, S.P.; Harrington, C.L.; Schumacher, K.; Hoffmann, T.; Tang, Y.Y.; Grill, E.; Schroeder, J.I. A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 2001, 411, 1053–1057, doi:10.1038/35082575. 11429606
- Sun, J.; Miwa, H.; Downie, J.A.; Oldroyd, G.E. Mastoparan activates calcium spiking analogous to Nod factor-induced responses in Medicago truncatula root hair cells. Plant Physiol. 2007, 144, 695–702, doi:10.1104/pp.106.093294. 17322338
- Miwa, H.; Sun, J.; Oldroyd, G.E.; Downie, J.A. Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus. Mol. Plant-Microbe Interact. 2006, 19, 914–923, doi:10.1094/MPMI-19-0914. 16903357
- Kosuta, S.; Hazledine, S.; Sun, J.; Miwa, H.; Morris, R.J.; Downie, J.A.; Oldroyd, G.E. Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes. Proc. Natl. Acad. Sci. USA 2008, 105, 9823–9828, doi:10.1073/pnas.0803499105. 18606999
- Nagai, T.; Yamada, S.; Tominaga, T.; Ichikawa, M.; Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 2004, 101, 10554–10559, doi:10.1073/pnas.0400417101. 15247428
- Monshausen, G.B.; Bibikova, T.N.; Messerli, M.A.; Shi, C.; Gilroy, S. Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs. Proc. Natl. Acad. Sci. USA 2007, 104, 20996–21001, doi:10.1073/pnas.0708586104. 18079291
- Monshausen, G.B.; Messerli, M.A.; Gilroy, S. Imaging of the Yellow Cameleon 36 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis. Plant Physiol. 2008, 147, 1690–1698, doi:10.1104/pp.108.123638. 18583529
- Verrillo, F.; Occhipinti, A.; Kanchiswamy, C.N.; Maffei, M.E. Quantitative analysis of herbivore-induced cytosolic calcium by using a Cameleon (YC 36) calcium sensor in Arabidopsis thaliana. J. Plant Physiol. 2014, 171, 136–139, doi:10.1016/j.jplph.2013.09.020. 24331428
- Behera, S.; Kudla, J. High-resolution imaging of cytoplasmic Ca2+ dynamics in Arabidopsis roots. Cold Spring Harb. Protoc. 2013, 2013, doi:10.1101/pdb.prot073023.
- Behera, S.; Kudla, J. Live cell imaging of cytoplasmic Ca2+ dynamics in Arabidopsis guard cells. Cold Spring Harb. Protoc. 2013, 2013, 665–669. 23818665
- Monshausen, G.B.; Bibikova, T.N.; Weisenseel, M.H.; Gilroy, S. Ca2+ regulates reactive oxygen species production and pH during mechanosensing in Arabidopsis roots. Plant Cell 2009, 21, 2341–2356, doi:10.1105/tpc.109.068395. 19654264
- Gjetting, S.K.; Schulz, A.; Fuglsang, A.T. Perspectives for using genetically encoded fluorescent biosensors in plants. Front. Plant Sci. 2013, 4, 234. 23874345
- Allen, G.J.; Murata, Y.; Chu, S.P.; Nafisi, M.; Schroeder, J.I. Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era1-2. Plant Cell 2002, 14, 1649–1662, doi:10.1105/tpc.010448. 12119381
- Allen, G.J.; Chu, S.P.; Schumacher, K.; Shimazaki, C.T.; Vafeados, D.; Kemper, A.; Hawke, S.D.; Tallman, G.; Tsien, R.Y.; Harper, J.F.; et al. Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant. Science 2000, 289, 2338–2342, doi:10.1126/science.289.5488.2338. 11009417
- Allen, G.J.; Kuchitsu, K.; Chu, S.P.; Murata, Y.; Schroeder, J.I. Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells. Plant Cell 1999, 11, 1785–1798. 10488243
- Sieberer, B.J.; Chabaud, M.; Timmers, A.C.; Monin, A.; Fournier, J.; Barker, D.G. A nuclear-targeted cameleon demonstrates intranuclear Ca2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors. Plant Physiol. 2009, 151, 1197–1206, doi:10.1104/pp.109.142851. 19700563
- Iwano, M.; Shiba, H.; Miwa, T.; Che, F.S.; Takayama, S.; Nagai, T.; Miyawaki, A.; Isogai, A. Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol. 2004, 136, 3562–3571, doi:10.1104/pp.104.046961. 15489279
- Miwa, H.; Sun, J.; Oldroyd, G.E.; Downie, J.A. Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell. Plant J. 2006, 48, 883–894, doi:10.1111/j.1365-313X.2006.02926.x. 17227545
- Richter, G.L.; Monshausen, G.B.; Krol, A.; Gilroy, S. Mechanical stimuli modulate lateral root organogenesis. Plant Physiol. 2009, 151, 1855–1866, doi:10.1104/pp.109.142448. 19794120
- Rincon-Zachary, M.; Teaster, N.D.; Sparks, J.A.; Valster, A.H.; Motes, C.M.; Blancaflor, E.B. Fluorescence resonance energy transfer-sensitized emission of yellow cameleon 360 reveals root zone-specific calcium signatures in Arabidopsis in response to aluminum and other trivalent cations. Plant Physiol. 2010, 152, 1442–1458, doi:10.1104/pp.109.147256. 20053711
- Palmer, A.E.; Giacomello, M.; Kortemme, T.; Hires, S.A.; Lev-Ram, V.; Baker, D.; Tsien, R.Y. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 2006, 13, 521–530, doi:10.1016/j.chembiol.2006.03.007. 16720273
- Patterson, G.H.; Knobel, S.M.; Sharif, W.D.; Kain, S.R.; Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 1997, 73, 2782–2790, doi:10.1016/S0006-3495(97)78307-3. 9370472
|Table 1. Main calcium indicators used in plant science.|
|Ca2+ indicator||Chemical structure||Properties||References|
|FLUO-3||The most important properties of Fluo-3 are an absorption spectrum compatible with excitation at 488 nm by argon-ion laser sources and a very large fluorescence intensity increase in response to Ca2+ binding.||[49,51–53]|
|FLUO-4||Fluo-4 and its esterified form Fluo4-AM, has a visible wavelength excitation (compatible with argon-ion laser sources) and a large fluorescence increase upon binding Ca2+.|||
|FLUO-4FF, FLUO-5F, FLUO-5N||These are analogs of Fluo-4 with lower Ca2+- binding affinity, making them suitable for detecting intracellular calcium levels in the 1 μM to 1 mM range that would saturate the response of Fluo-3 and Fluo-4|||
|FLUO-4 DEXTRANS||These are Fluo-4 coupled to a biologically inert dextran carrier (molecular weight = 10,000), providing a new and potentially valuable tool for measuring Ca2+ transients.|||
|CALCIUM GREEN™-1||Structurally similar to Fluo-3, but is more fluorescent at low calcium concentrations, facilitating the determination of base line Ca2+ levels and increasing the visibility of resting cells||[56,57]|
|CALCIUM GREEN™-2||It has two fluorescent reporter groups, which are believed to quench one another in the absence of calcium, that undergo a much larger increase in fluorescence emission upon calcium binding than does Calcium Green™-1. Its lower affinity for calcium makes it particularly suited to measuring relatively high spikes of calcium, up to 25 μM.|||
|CALCIUM ORANGE™||These are spectrally similar to tetramethylrhodamine and Texas Red. The long-wavelength spectral characteristics of these indicators allow them to be used in combination with fluorescein and ultraviolet excitable dyes.||[37,44,50,59]|
|INDO-1||These are “dual-emission” and “dual-excitation” types of calcium dyes, respectively. To utilize either of these indicators, however, appropriate modifications of standard CLSM need to be made, such as a high-power argon-ion laser is required to obtain ultraviolet (UV) excitation, and compensatory changes along the optical path must be incorporated to deal with the lens aberrations and reduced signal throughputs that are associated with UV illuminations. At low concentrations of the indicator, use of the 340/380 nm excitation ratio for Fura-2 or the 405/485 nm emission ratio for Indo-1 allows accurate measurements of the intracellular Ca2+ concentration. Measurements of Indo-1 and Fura-2 fluorescence can usually be made over a period of an hour without significant loss of fluorescence resulting from either leakage or bleaching. In addition, Fura-2 and Indo-1 are bright enough to permit measurements at intracellular concentrations of dye unlikely to cause significant Ca2+ buffering or damping of Ca2+ transients.||[11,46,60]|
|RHODAMINE-BASED INDICATORS.||Rhod-2 has fluorescence excitation and emission maxima at 552 and 581 nm, respectively. Variants with longer-wave length excitation and emission (X-Rhod-1) and lower Ca2+-binding affinity (Rhod-5N, Rhod-FF, etc.) have been developed (i.e., at Molecular Probes). Rhod-2 is used as a selective indicator for mitochondrial Ca2+ in most eukaryotic cells.|||
|Table 2. Main GFP-based Ca2+ indicators used in plant science.|
|YC2.1||Suitable to use under various physiological condition because of their lower sensitivity to pH.||Ca2+ fluxes in Arabidopsis guard cells, Ca2+ elicitation in NOD factor of medicago tranculata, Ca2+ role in plant interaction with symbiotic bacteria (rhizobium) and fungal (mycorrhizal) root colonization.||[27,33,82–85,93–96]|
|Nucleoplasmin- YC2.1||Suitable for nuclear matrix localization study||Sieberer and colleague showed that Ca2+ spiking localized to nuclear matrix in the root hairs in response to external nodulation factors.|||
|D3cpv||Suitable for studying peroxisomal Ca2+ dynamics||Costa and colleagues showed peroxisomal Ca2+ dynamics under stress signaling.|||
|YC4.6||Suitable for studying endoplasmic reticulum Ca2+ dynamics||Iwano and colleagues showed pollen tube endoplasmic reticulum Ca2+ dynamics|||
|YC3.6||Substitution of acceptor yellow fluorescent protein yielded five fold increased signal sensitivity, which allowed imaging of both temporal and dynamic signaling of cytosolic Ca2+ fluxes.||Used to study Ca2+ dynamics ranging from roots and root hairs, guard cells, pollen and leaves upon mechanical and herbivore damage.||[19,27,61,79,82,84, 86–89,92,94,96,98–101]|
© 2014 by the authors; licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).