Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Summary

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.

Abstract

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.

Introduction

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 stress^2-4.

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 cytosol^14,15, nucleus¹⁶, mitochondria¹⁷, and plasma membrane¹⁸, and YC4.6 and D4ER were targeted to the ER^15,19, 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’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 stress^4,21 . 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.

Protocol

1. Aequorin Based Ca²⁺ Imaging Using the FAS System

1. 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 °C for 2 days (Figure 1A).
2. 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)
3. Incubate the seedlings with cofactor. Place the adhered seedlings onto the square plate (10 x 10 cm) containing 15 ml of 2 μg/ml h-CTZ (coelenterazine) in water. Incubate the seedlings at room temperature for 4 hr to overnight (Figure 1F).
4. 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.
5. 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.
6. 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).

2. Live Cell Confocal Ca²⁺ Imaging

1. 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 °C for 2 days.
2. Setup Imaging Chambers
   1. 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).
   2. Assemble an imaging chamber using a plexiglass chamber (Chamber B).
      1. 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).
      2. 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).
   3. 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).
3. Acquire images using a confocal microscope. To apply stress stimuli, such as salt, cold or peroxide, slowly inject about 200 μl 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 μl or 100 μl 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.
4. 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.

Results

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 stimuli we examined in this study, 75 mM NaCl induced the strongest Ca²⁺ response in leaves and roots during the first 40 sec of exposure (Figure 2A middle panel and Figure 2B), while 1 mM H₂O₂ induced a strong Ca²⁺ response both in leaves and roots during the first and second 40 sec (Figure 2A right panel). The solution of 400 mM mannitol only induced Ca²⁺ response in roots during the first 40s and very weak signals in leaves during the second 40 sec. Cu²⁺ strongly enhanced the amplitudes and duration of Ca²⁺ signals triggered by NaCl and mannitol (Figure 2A left panel). It appears that Cu²⁺ had inhibitory effects on H₂O₂ induced Ca²⁺ signals, reducing both the amplitude and duration (Figure 2A right panel and 2B).

Using Case12 expressing Arabidopsis plants, we measured Ca²⁺ dynamics in the cytosol and nuclei simultaneously. Molecular weight of Case12 is 46 kDa, therefore transgenic plants expressing Case12 have GFP signals in both the cytosol and nuclei of leaf and root cells (Figure 4A). Customized (Figure 3A and 3B) or commercial chambers (Figure 3C) were used for live cell imaging with a confocal microscope. Applying 150 mM NaCl to the seedling in the chamber, we observed changes of fluorescence intensity in both the cytosol and nucleus of the individual cells (Figure 4B and C; Supplementary Movie 1). Using NIS-Elements (Imaging software, Nikon Instruments Inc.), we measured the fluorescence intensity in regions of interest (ROI) over time. The amplitude, duration and frequency of cytosolic and nuclear Ca²⁺ oscillation are presented graphically in Figure 4B. The amplitude and period of Ca²⁺ spikes in the cytoplasm and the nuclei vary greatly between cells of the same root, as well as across cells in different roots. The amplitude and period of cytosolic Ca²⁺ spikes were 60.36 ± 45.22 AU (arbitrary unit) and 58.24 ± 15.70 sec, respectively. The amplitude and period of nuclear Ca²⁺ spikes were 316.26 ± 75.24 (AU) and 61.71 ± 16.31 sec, respectively. In contrast, the duration of cytosolic and nuclear Ca²⁺ spikes were similar between cells, which were 17.43 ± 3.67 sec and 17.33 ± 2 sec, respectively. The amplitude of Ca²⁺ spikes decreased over time, which may be due to the change of the focal plane and/ or a loss of response to stress stimuli because of an unfavorable in vitro condition. These results show that salt stress triggers Ca²⁺ oscillation in both the cytoplasm and nucleus.

Figure 1. Aequorin based FAS system for measuring spatial-temporal Ca²⁺ dynamics in response to stress stimuli. A) Grow Arabidopsis seedlings vertically on a plate containing MS media. B, C) Place an adhesive film (B) on the top of seedlings (C). D, E) Transfer 10 day old seedlings onto the adhesive film. F) Incubate seedlings adhered to the film with 2 µg/ml of h-CTZ in water for 4 hr. G) Cut the film down the middle and place each piece into a different empty plate. Leave the plates in the dark for 5 min. Apply simultaneously control and stimuli solutions (be sure the seedlings are entirely covered), and acquire images immediately. The scale bar is 5 cm.

Figure 2. Comparison of spatial-temporal Ca²⁺ response of 10 day seedlings to different stress stimuli. A) A time series of luminescence images of seedlings subjected to 400 mM mannitol and 400 mM mannitol plus 1 mM CuCl₂ (A, left panel), 75 mM NaCl and 75 mM NaCl plus 1 mM CuCl₂ (A, middle panel), and 1 mM H₂O₂ or 1 mM H₂O₂ plus 1 mM CuCl₂ (A, right panel). Upper and lower panels of A are luminescence images acquired during first 40 sec and second 40 sec, respectively. An intensity scale bar indicates an increase of luminescence signals from low (black) to high (white). B) Bar chart of average luminescence intensity of seedlings in response to the indicated stress stimuli.

Figure 3. Setup of a live cell confocal imaging experiment. A) A customized chamber is built with a slide, coverslip with four pieces of clay and cotton wool. Two 5 day old seedlings are placed on the slide. One end of a polyethylene tube is placed next to the assembled chamber and fixed with a pieces of tape while the other end is connected to a 1 ml syringe. B) A customized slide made with plexiglass has a open chamber in the middle and two channels that are connected to the two sides of channel. One coverslip is placed on the top of the slide and two polyethlene tubes are placed in the channel. Coverslip and tubes are fixed in the position with silicone grease. The end of one of the tubes is connected to a 1 ml syringe and the end of another tube remains open. A 5 day old seedling is placed in the chamber and a coveslip is put on the top of the seedling and sealed with silicone grease. The plexigalss slide with the assembled chamber is placed on the stage of the microscope. C) Arabidopsis seedlings are grown in two wells (left) or 8 wells (right) of the chambered cover glass containing a thin layer of 0.6% of phytagel in MS media.

Figure 4. Cytosolic and nuclear Ca²⁺ oscillation in response to salt stress. A) Confocal images show that Case12 is expressed in the cytoplasm and nuclei of root cells (left panel) and leaf cells (right panel) of transgenic plants expressing Case12. The image on the right panel is a merged image of red autofluorescence of the chloroplasts and green fluorescence of Case12. B) Salt stress (150 mM NaCl) induced Ca²⁺ oscillation in the cytosol (upper panel) and nuclei (lower panel) of root cells. Locations of the measured cells are indicated by arrows with numbers on panel.

Discussion

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 of FAS allows the use of low intensity stimuli for examining Ca²⁺ responses, which is important for avoiding artifacts caused by physical damages to cells when high intensity stress is used. Seven to 10 day old Arabidopsis seedlings showed tissue or stimuli-specific Ca²⁺ response, though seedlings at different physiological stages may have different sensitivity and specificity in response to a given stimulus. Therefore it is preferable to use 7-10 day old seedlings to increase the throughput of FAS measurements, in which around 100 seedlings could be adhered onto one piece of film. Together with easy and simple performance, high sensitivity and reproducibility, the FAS system could also be adapted for screening EMS mutagenized mutants for altered Ca²⁺ responses. For screening purposes, it is crucial to have uniform seedlings that are well grown on the plate and adhered onto the film. Four to five hours incubation of film adhered seedlings with h-CTZ is usually sufficient for reconstitution of aequorin. Using the adhesive film with holes is preferable because reaction of apoaequorin and cofactor requires oxygen. For the comparison of tissue or stimuli-specific Ca²⁺ responses, using groups of seedlings originally from the same film is critical because the state of seedlings and/or the reconstitution condition may affect the total amount of functional aequorin. When these requirements are met, the difference of luminescence response is proportional to the tissue- or stimulus-specific Ca²⁺ response.

Aequorin based measurement of Ca²⁺ signals reflect the response of a population of the cells. However, considering the heterogeneity of cell types and/or the different accessibility of cells in the tissues to the stimuli, the results of Aequorin based Ca²⁺ measurements do not equate to the behavior of any one cell. Understanding Ca²⁺ dynamics at the cellular level requires a Ca²⁺ indicator that can be detected with subcellular resolution. Live cell confocal imaging of fluorescence protein (FP)-based Ca²⁺ indicators provides an advanced Ca²⁺ detection system for recording cellular Ca²⁺ dynamics with cellular resolution in a desired tissue. Here, using live cell confocal imaging and FP-based Ca²⁺ indicators, we recorded subcellular Ca²⁺ transients in both the cytoplasm and nuclei simultaneously. Two types of FP-based Ca²⁺ indicators have been implemented in plants with different characteristics. Compared to YCs, single GFP-based Ca²⁺ indicator has some advantages: 1. Case12 does not require YFP and CFP filter sets or the many assumptions and complex calculations needed to measure FRET²². Case12 has single excitation/emission maxima and can be easily detected using standard GFP filter set. 2. Case12 is stable under physiological pH, and is a relatively a small protein (46 kDa). When expressed in plants, non-targeted Case12 appears in the cytoplasm as well as the nuclei, so Case12 transgenic plants can be used to track cytosolic and nuclear Ca²⁺ dynamics simultaneously. A drawback of this reporter is that it is a non-ratiometric indicator, so fluorescence levels are also affected by factors that are unrelated to Ca²⁺ concentration, such as the expression level of Case12 and local pH. Nevertheless, Case12 is adequate to determine cellular Ca²⁺ concentration for the comparison of spatial-temporal Ca²⁺ dynamics in the cytoplasm and organelles between materials with different genetic backgrounds under the same experimental conditions. In the future, new GFP based Ca²⁺ indicators with higher signal-to-noise ratio, fast kinetics and greater response, like GCaMP3²⁴ and GCaMP5²⁵ demonstrated in animals, could be better indicators for recording cellular or subcelluar Ca²⁺ dynamics in plants.

The Aequorin and Case12 detection systems described here provide nondestructive in vivo approaches for the measurement of spatial-temporal Ca²⁺ dynamics at the whole seedling and subcellular levels, respectively. In both systems, the physiological state or growth conditions could affect the Ca²⁺ responses of the seedlings or cells. If physically damaged or not grown in a good condition, cells could have weak or even no Ca²⁺ responses. In addition, the duration of spatial and temporal Ca²⁺ signals are also affected by the conditions of the seedling during the measurements. The luminescence or fluorescence signals may decrease over time due to stimuli-induced damage, and/or the laser-induced photo-damage in the case of confocal imaging. Although stimulus-induced damage cannot be avoided, phototoxicity can be reduced by limiting the intensity and/or frequency of laser exposure. In addition, Aequorin based Ca²⁺ recording system requires a reconstitution step. Factors that interrupt the diffusion of CTZ to the cell and/or reaction of apoaequorin with CTZ would affect luminescence signals that are related directly to Ca²⁺ concentration. Therefore the concentration of CTZ, as well as the duration and conditions of CTZ incubation need to be optimized for best sensitivity and reproducibility. Genetically encoded Ca²⁺ indicators may inaccurately report cellular Ca²⁺ concentrations for other reasons, including unintended targeting to specific subcellular compartments, temperature sensitivity, or interference of the reporter with the cellular Ca2+ signaling machinery²³. We found that Aequorin or Case12 expressing plants have no obvious morphological or conditional phenotypes such as altered stress tolerance. Therefore it is unlikely that these genetically encoded Ca²⁺ indicators have major interferences with Ca²⁺ signaling in plants.

In summary, we present here detailed instructions for the use of two Ca²⁺ detection systems for measuring spatial and temporal Ca²⁺ signals at both the whole plant seedling and subcellular levels. These two systems can be used to determine tissue (or cell type) or stimuli-specific Ca²⁺ dynamic patterns with high sensitivity and reproducibility. Therefore they are powerful tools for elucidating the gene networks that underlie Ca²⁺ signaling in response to various environmental cues.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm x 10 cm square Petri dish	VWR	60872-310
Adhesive film	VWR	60941-062
Polyethylene tubing	PerkinElmer	9908265
1 ml syringe	VWR	53548-000
Silicone grease	Beckman	335148
2-well chambered cover glass	Nalge Nunc international	155379
8-well chambered cover glass	Nalge Nunc international	155409
Luminescence imaging system	Princeton Instruments	N/A
Inverted confocal laser-scanning microscope	Nikon Instruments Inc.	N/A	Nikon A1R
Imaging software	Nikon Instruments Inc.	N/A	Nikon Elements
DataGraph	Visual Data Tools Inc	N/A	DataGraph 3.1.1 is the newest version
Coelenterazine	NanoLight Technolgies	#301B NF-BCTZ-FB
All purpose bleach	Any local store	N/A
Triton X-100	Fisher	BP151500
MS salt	Phytotechnology Labs	M524-50L
Sucrose	VWR	BDH8029-12KG
Agar	Sigma	A1296-5KG
Phytagel	Sigma	P8169-1KG