We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1. Aequorin Based Ca2+ Imaging Using the FAS System
<ol>
	<li>Prepare seedlings for luminescence imaging. Sterilize seeds of Arabidopsis plants expressing Aequorin with 10% bleach solution containing 0.01% Triton-100. Sow the sterile seeds on a square plate (10 x 10 cm square Petri dish with grid,) containing full strength MS (Murashige and Skoog Basal Salt Mixture), 1% sucrose, and 1.2% agar. Place plates vertically in a growth chamber after stratification at 4 &#176;C for 2&#160;days (Figure 1A).</li>
	<li>Transfer the seedlings onto a film. Place an adhesive film (Figure 1B) on the top of 7-10 day old seedlings growing on the plate. Gently push the film by hand to ensure that seedlings adhere to the film (Figure 1C). Peel the film gently so that the seedlings remain adhered to the film (Figure 1D and 1E)</li>
	<li>Incubate the seedlings with cofactor. Place the adhered seedlings onto the square plate (10 x 10 cm) containing 15 ml of 2 &#956;g/ml h-CTZ (coelenterazine) in water. Incubate the seedlings at room temperature for 4 hr to overnight (Figure 1F).</li>
	<li>Prepare for luminescence imaging. Take the film out of the h-CTZ solution and cut it down the middle, forming two pieces. Place each piece of film with seedlings face up in two different plates. Leave the plates in the dark for 5 min.</li>
	<li>Acquire luminescence images. In the dark, place the two plates next to each other on the stage of the luminescence imaging system (Figure 1G). Acquire images immediately upon adding 20 ml of stimuli solution to the plates simultaneously.</li>
	<li>Analyze luminescence images. Choose the same display range for all luminescence images. Crop the region of interest (ROI) and generate the images as JPEG files (Figure 2A). Alternatively, export the images as SPE format files and import them into the ImageJ image analysis software. Set measurements for calculation of the mean gray value of ROI. Select the same size of ROI area and measure the mean gray and present data as bar graphs (Figure 2B).</li>
</ol>
2. Live Cell Confocal Ca2+&#160;Imaging
<ol>
	<li>Prepare seedlings for confocal imaging. Sterilize seeds of Arabidopsis plants expressing case12 with 10% bleach solution containing 0.01% Triton. Sow the sterile seeds on a plate containing full- strength MS salts, 1% sucrose, and 1.2% agar. Place plates vertically in a growth chamber after stratification at 4 &#176;C for 2&#160;days.</li>
	<li>Setup Imaging Chambers
	<ol>
		<li>Assemble an imaging chamber using a slide and coverslip (Chamber A). Place a piece of water soaked cotton wool onto the middle of the slide. Then transfer one or two 5 day old seedlings from the plate onto the top of water soaked cotton wool. Stick small pieces of clay to each corner of a coverslip, and place coverslip on the top of seedlings to create a gap (chamber) between the coverslip and slide (Figure 3A, left panel). Connect one end of polyethylene tube (0.58 mm diameter) to a 1 ml syringe and place the other end of tube immediately adjacent to the chamber. Hold the tubing in place with tape (Figure 3A, right panel).</li>
		<li>Assemble an imaging chamber using a plexiglass chamber (Chamber B).
		<ol>
			<li>Spread a thin layer of silicone grease around two polyethylene tubes (0.58 mm diameter) and press the coated tubes into the channels on each side of the plexiglass chamber (Figure 3B left panel). Spread a thin layer of silicone grease onto the surface of the chamber that contains the tube grooves and press a coverslip into the grease to seal one side of the chamber opening (cut out).</li>
			<li>Transfer a five day old seedling from the plate into the chamber and place a piece of cotton wool soaked with water on the top of the seedling. Spread silicone grease onto the other surface of the chamber, and press another coverslip on top to seal. Connect the end of one of the tubes to a 1 ml syringe. Leave the end of the other tube open (Figure 3B right panel).</li>
		</ol>
		</li>
		<li>Prepare an imaging chamber using a chambered cover glass (Chamber C). Sterilize the chambered cover glass with 70% ethanol and leave it on the hood until dry. Add 0.6 ml or 0.2 ml of full strength MS medium containing 1% sucrose and 0.5% phytagel into each well of an 2-well chamber or an 8-well chamber (Figure 3C right panel), respectively. Sterilize seeds and sow the seeds directly in a chambered cover glass containing a thin layer of clear gel and let them grow vertically for 5 days (Figure 3C).</li>
	</ol>
	</li>
	<li>Acquire images using a confocal microscope. To apply stress stimuli, such as salt, cold or peroxide, slowly inject about 200 &#956;l of 150 mM NaCl, ice-cold water or 1 mM H2O2 solution into the chamber (Chamber A or B) just prior to acquiring images, or gently add 500 &#956;l or 100 &#956;l of stimulus solution to the well of a 2- or 8-well chamber, respectively. Capture images immediately after applying the stimulus solution using an inverted Nikon A1R confocal laser-scanning microscope with a 20X water immersion lens (numerical aperture 0.75). Collect a time series of images at 4 sec intervals with excitation and emission wavelengths of 488 nm and 500 - 550 nm, respectively, and at a pixel resolution of 512 x 512.</li>
	<li>Image Analysis 
	Using Nikon Elements, ROIs were drawn around each cell (or area) of interest. Total intensity within each ROI was measured over time using the Time Measurement dialog box (ImageJ could be used instead). Total intensity measurements were exported and processed in DataGraph. Ca2+ spike amplitude was defined as peak intensity minus resting intensity, duration as the time between initiation and completion of a spike, and period as the time interval between adjacent spike peaks of two spikes. t-test was used to compare the means.</li>
</ol>

<ol>
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Genomics. 39 (5), 225-235 (2012).</li><li>Ranf, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense-related+calcium+signaling+mutants+uncovered+via+a+quantitative+high-throughput+screen+in+Arabidopsis+thaliana.">Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana.</a> Mol. Plant. 5 (1), 115-130 (2012).</li><li>Zhu, X., Feng, Y., Liang, G., Liu, N., Zhu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin-based+luminescence+imaging+reveals+stimulus-+and+tissue-specific+Ca2++dynamics+in+Arabidopsis+plants.">Aequorin-based luminescence imaging reveals stimulus- and tissue-specific Ca2+ dynamics in Arabidopsis plants.</a> Mol. Plant. 6 (2), 444-455 (2013).</li><li>Knight, M. R., Campbell, A. K., Smith, S. 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The authors have nothing to disclose.

We have demonstrated a FAS system for recording the spatial-temporal Ca2+ response of Arabidopsis seedlings. This FAS Ca2+ recording system provides a simple, sensitive and robust approach that could be adapted for measuring Ca2+ dynamics triggered by various stimuli in addition to the abiotic stress stimuli that are presented here. Using this system, we can easily compare tissue- or stimuli-specific spatial-temporal Ca2+ dynamics at the whole plant level. The high sensitivity ...

The plant cell responds to the environment via signaling that coordinates cell actions. An early cell signaling event in response to environmental stimuli is a transient Ca2+ increase. The pattern, or signature of a transient increase in free Ca2+ concentration is characterized by its amplitude, frequency, and duration. Distinct spatio-temporal Ca2+ signatures regulate different cellular activities1. Specific stimuli, such as heat, cold, salt, drought, light, or plant hormones, may fine-tune the spatio-temporal activity of membrane-localized Ca2+ channels and/or transporters, resulting in specific Ca2+ signatures. Although Ca2+ transporters have been well characterized, little is known about the molecular identities and functions of Ca2+ channels in plants1. Genetic screens for mutants with altered Ca2+ response to stress stimuli may be an effective approach for identifying the components that compose Ca2+ signatures. Recently several Aequorin based Ca2+ detection systems have been developed that facilitate genetic screens for Ca2+ signaling components in response to pathogen attack and abiotic stress2-4.
Aequorin was first used to detect Ca2+ signals in plants in the early 1990s5. Since then, Aequorin has been targeted to different cellular compartments, such as the cytoplasm5, nucleus6, chloroplasts7, tonoplast8, mitochondria9, and stroma10, as well as to different cell types in the root to monitor cell specific Ca2+ signals11. Aequorin based Ca2+ measurements reveal the spatial and temporal Ca2+ response of a population of cells to stress stimuli. However, in most cases, the Ca2+ responses of single cells are unsynchronized in the responding tissue4. Therefore, Aequorin Ca2+ recording does not necessarily report the Ca2+ signal in individual cells. In recent years, genetically encoded fluorescent protein (FP)-based Ca2+ indicators, such as yellow cameleon (YCs)12 and CASEs1213 have been used to study Ca2+ signaling with high subcellular resolution. YCs are fluorescence resonance energy transfer (FRET)-based Ca2+ indicators, containing CFP and YFP variants linked by the Ca2+-binding protein calmodulin and calmodulin-binding peptide M13. Calmodulin undergoes a conformational change as it binds to Ca2+, thereby brings CFP and YFP closer together, resulting in increased energy transfer (enhanced FRET). The FRET level over time, calculated roughly as the ratio of YFP to CFP signal intensities, reflects intracellular Ca2+ dynamics. Several YC versions have been used in plants. YC3.6 was targeted to the cytosol14,15, nucleus16, mitochondria17, and plasma membrane18, and YC4.6 and D4ER were targeted to the ER15,19, and D3cpv was targeted to the peroxisomes20. Transgenic plants expressing YCs allow the live-cell imaging of Ca2+ dynamics within different cellular compartments of different cell types. CASEs (presumably Calcium sensor) are single circularly permuted fluorescent proteins (cpFPs) harboring a calmodulin and calmodulin-binding peptide M13. Upon binding to Ca2+, CASEs undergo conformational changes, leading to an increase of fluorescence intensity. The correlation between the CASE&#8217;s fluorescence response and Ca2+ concentration allows intracellular Ca2+ dynamics to be measured quantitatively. The Case12 variant has 12 fold increased fluorescence in the Ca2+-saturated forms. N. benthiminana plants transiently expressing Case12 or Arabidopsis plants stably expressing Case 12 were used to study Ca2+ signaling in defense and abiotic stress4,21 . Asynchronous spatial and temporal Ca2+ oscillations in cells responding to pathogen attack, or to dehydration stress have been revealed with Case12 based Ca2+ imaging.
Here, we present detailed instructions for Aequorin based luminescence imaging of tissue- and stimuli specific Ca2+ dynamics in Arabidopsis seedlings, and for confocal imaging of cytosolic and nuclear Ca2+ dynamics in Arabidopsis root cells that express Case 12. Luminescence imaging of FAS could be adapted to analyze stress-induced Ca2+ dynamics in intact plants or tissues not described here, or to screen mutagenized Arabidopsis plant populations for mutants with altered stress induced Ca2+ signals. The live cell Ca2+ imaging setup could be adapted to analyze Ca2+ dynamics within different subcelluar compartments or in different cell types using other Ca2+ indicators.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X10 cm square petri dish&#160;</td>
			<td>VWR</td>
			<td style="width:175px;">60872-310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">adhesive film&#160;</td>
			<td style="width:172px;">VWR</td>
			<td>60941-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylene tubing</td>
			<td>PerkinElmer</td>
			<td style="width:175px;">9908265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL syringe&#160;</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">53548-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silicone grease&#160;</td>
			<td style="width:172px;">Beckman</td>
			<td style="width:175px;">335148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well chambered cover glass&#160;</td>
			<td>Nalge Nunc international</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">8-well chambered cover glass</td>
			<td>Nalge Nunc international</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Imaging software</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td>Nikon Elements&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">ImageJ</td>
			<td style="width:172px;">NIH</td>
			<td style="width:175px;">N/A</td>
			<td>Public domain, Java-based image processing program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DataGraph</td>
			<td>Visual Data Tools Inc</td>
			<td style="width:175px;">N/A</td>
			<td>DataGraph 3.1.1 is the newest version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coelenterazine</td>
			<td>NanoLight Technolgies</td>
			<td style="width:175px;">#301B NF-BCTZ-FB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">All purpose Bleach</td>
			<td style="width:172px;">Any local store</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Triton X-100</td>
			<td style="width:172px;">Fisher</td>
			<td style="width:175px;">BP151500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">MS salt</td>
			<td style="width:172px;">Phytotechnology Labs</td>
			<td style="width:175px;">M524-50L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">sucrose</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">BDH8029-12KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">A1296-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Phytagel</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">P8169-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spatial and temporal ca2+ signals in arabidopsis plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Mannitol, NaCl and H2O2 were used as proxies for dehydration, salt and oxidative stress stimuli, respectively. To check if the heavy metal ion Cu2+ synergizes with any of these three stress stimuli, we compared the Ca2+ response to each stimuli in the presence or absence of Cu2+. As shown in Figure 2, FAS luminescence imaging revealed that Arabidoposis seedlings responded differently to dehydration, salt and oxidative stress. For the concentration of...

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Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

12.0K Views.  Purdue University. Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.